Beta-1,3-galactosyltransferase homologs

ABSTRACT

The present invention relates to polynucleotide and polypeptide molecules for znssp2, a novel member of the galactosyltransferase family. The polypeptides, and polynucleotides encoding them, are cell-cell interaction and glycoprotein synthesis modulating and may be used for delivery and therapeutics. The present invention also includes antibodies to the znssp2 polypeptides.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to Provisional Application No.60/111,697 filed on Dec. 10, 1998. Under 35 U.S.C. §119(e)(1), thisapplication claims benefit of said Provisional Application.

BACKGROUND OF THE INVENTION

[0002] Beta-1,3-galactosyltransferase molecules are classified in thefamily of glycosyltransferases. In addition to transferring carbohydratemolecules to glycoproteins during biosynthesis, members of this familyhave also been detected on the cell surface where they are thought to beinvolved in varying aspects of cell-cell interactions. This familyincludes carbohydrate transferring enzymes, such as sialyltransferasesand fucosyltransferases, and galactosyltransferases. During theformation of O-linked glycoproteins and the modification of N-linkedones, each sugar transfer is catalyzed by a different type ofglycosyltransferase. Each glycosyltransferase enzyme is specific forboth the donor sugar nucleotide and the acceptor molecule.

[0003] Galactosyltransferases promote the transfer of an activatedgalactose residue in UDP-galactose to the monosaccharideN-acetylglucosamine. This transfer is a step in the biosynthesis of thecarbohydrate portion of galactose-containing glycoproteins, such asoligosaccharides and glycolipids, in animal tissues. TheBeta-1,3-galactosyl-transferases are characterized by the elongation oftype I oligosaccharide chains, and the Beta-1,4-galactosyl-transferasesare characterized by the elongation of type II oligosaccharide chains.Both types of carbohydrate structures are present in solubleoligosaccharides of human milk, and are also found on glycoproteins andglycolipids, and are important precursors of blood group antigens. Bothgalactosyltransferases require a divalent cation (Mn²⁺) to function.Beta-1,4-galactosyltransferases are expressed in various cell types andtissues, while the Beta-1,3-galactosyltransferases seem to have morerestricted tissue distributions.

[0004] Some galactosyltransferases are found in the Golgi apparatus.These Golgi-localized enzymes have structure similarity: a shortN-terminal domain that faces the cytosol, a single transmembrane ahelix, and a large C-terminal domain that faces the Golgi lumen and thatcontains the catalytic site. The transmembrane α helix is necessary andsufficient to restrict the enzyme to the Golgi. Of theBeta-1,3-galactosyltransferase family two members (See Amado, M. et al.,J. Biol. Chem. 273, 21: 12770-12778, 1998) have been predicted to havetwo potentially different initiation codons, resulting in two differentN-terminal cytoplasmic domains.

[0005] Additionally, galactosyltransferases have been shown to beexpressed on the cell surface, where their function is theorized toparticipate in cellular interactions, perhaps as receptors, orreceptor-like complementary molecules. As a cell surface carbohydrate,galactosyltransferases have been implicated in varied biology such ascell migration, contact inhibition, tissue interactions, neuronalspecificity, fertilization, embryonic cell adhesions, limb budmorphogenesis, mesenchyme development, immune recognition, growthcontrol, and tumor metastasis. See, for example, Shur, B. D., Mol CellBioc. 61:143-158, 1984.

[0006] The failure of tumor cell-tumor cell adhesion is believed to be acontributing factor to tumor metastases. See, for example, Zetter,Cancer Biology, 4: 219-29, 1993. Metastases, in turn, are generallyassociated with poor prognosis for cancer treatment. The metastaticprocess involves a variety of cellular events, including angiogenesis,tumor cell invasion of the vascular or lymphatic circulation, tumor cellarrest at a secondary site; tumor cell passage across the vessel wallinto the parenchymal tissue, and tumor cell proliferation at thesecondary site. Thus, both positive and negative regulation of adhesionare necessary for metastasis. That is, tumor cells must break away fromthe primary tumor mass, travel in circulation and adhere to cellularand/or extracellular matrix elements at a secondary site. Moleculescapable of modulating cell-cell and cell-matrix adhesion are thereforesought for the study, diagnosis, prevention or treatment of metastases.

[0007] β1→3 Galactosyltransferases have limited homology to each other.In contrast to other glycosyltransferases, they do not appear to belocalized to the same chromosomes. Additionally, a member of this familyhas recently been identified in Drosophila. This molecule, Brainiac, isinvolved in contact and adhesion between germ-line and follicle cells(Amado, M. et al., J. Biol. Chem. 273, 21: 12770-12778, 1998).

[0008] A deficiency of Beta-1,3-galactosyltransferase enzymes has beennoticed in the Tn-syndrome. This syndrome is a rarely acquired disorderaffecting all hemopoietic lineages, and is characterized by theexpression of the Tn and the sialosyl-Tn antigens on the cell surface.The Tn is αN-acetylgalactosamine linked O-glycosidically to threonine orserine residues of membrane proteins. These antigens bind naturallyoccurring serum antibodies thereby leading to mild hemolytic anemia andpronounced thrombopenia. Thus, the blood cells in the Tn-syndrome areexpected to carry less sialic acid if galactose can not be transferredto N-Acetylgalactosamine. The expression of Tn and sialosyl-Tn antigensas a consequence of imcomplete or disordered gylcan biosynthesis hasbeen recognized as a cancer-associated phenomenon. Tn and sialosyl-Tnantigens are among the most investigated cancer-associated carbohydratesantigens.

[0009] The present invention provides such polypeptides for these andother uses that should be apparent to those skilled in the art from theteachings herein.

SUMMARY OF THE INVENTION

[0010] Within one aspect, the present invention provides an isolatedpolypeptide comprising residues 148 to 397 of SEQ ID NO:2. Within anembodiment, the isolated polypeptide comprises residues 19 to 397 of SEQID NO:2. Within another embodiment, the isolated polypeptide comprisesresidues 1 to 397 of SEQ ID NO:2.

[0011] Within another aspect, the present invention provides an isolatedpolypeptide selected from the group consisting of: a polypeptidecomprising residues 1 to 18 of SEQ ID NO:2; a polypeptide comprisingresidues 19 to 147 of SEQ ID NO:2; a polypeptide comprising residues 148to 397 of SEQ ID NO:2; a polypeptide comprising residues 19 to 397 ofSEQ ID NO:2; and a polypeptide comprising residues 1 to 397 of SEQ IDNO:2.

[0012] Within another aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide wherein the polypeptide comprisesresidues 148 to 397 of SEQ ID NO:2. Within an embodiment, thepolypeptide molecule comprises residues 19 to 397 of SEQ ID NO:2. Withinanothe embodiment, the polypeptide molecule comprises residues 1 to 397of SEQ ID NO:2.

[0013] Within another aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide molecule wherein the polypeptideis selected from the group consisting of: a polypeptide comprisingresidues 1 to 18 of SEQ ID NO:2; a polypeptide comprising residues 19 to147 of SEQ ID NO:2; a polypeptide comprising residues 148 to 397 of SEQID NO:2; a polypeptide comprising residues 19 to 397 of SEQ ID NO:2; anda polypeptide comprising residues 1 to 397 of SEQ ID NO:2. Within anembodiment is provided an expression vector comprising the followingoperably linked elements: a) a transcription promoter; b) a DNA segmentwherein the DNA segment is a polynucleotide encoding the polypeptide ofclaim 1; and a transcription terminator. Within another embodiment theDNA segment contains an affinity tag. Within another embodiment, theinvention provides a cultured cell into which has been introduced theexpression vector, wherein said cell expresses the polypeptide encodedby the DNA segment. Within another embodiment the invention provides amethod of producing a polypeptide comprising culturing the cell, wherebysaid cell expresses the polypeptide encoded by the DNA segment; andrecovering the polypeptide.

[0014] Within another aspect is provided a method of producing anantibody comprising the following steps in order: inoculating an animalwith a polypeptide selected from the group consisting of: a polypeptidecomprising residues 1 to 18 of SEQ ID NO:2; a polypeptide comprisingresidues 19 to 147 of SEQ ID NO:2; a polypeptide comprising residues 148to 397 of SEQ ID NO:2; a polypeptide comprising residues 19 to 397 ofSEQ ID NO:2; and a polypeptide comprising residues 1 to 397 of SEQ IDNO:2, wherein the polypeptide elicits an immune response in the animalto produce the antibody; and isolating the antibody from the animal.Within an embodiment the antibody produced binds to a residues 1 to 397of SEQ ID NO:2. Within another embodiment the antibody is a monoclonalantibody. Within another embodiment the antibody specifically binds to apolypeptide of residues 1 to 397 of SEQ ID NO:2.

[0015] Within another apsect is provided a method of producing anantibody comprising the following steps in order: inoculating an animalwith an epitope bearing portion of a polypeptide wherein the epitopebearing portion is selected from the group consisting of: a polypeptideconsisting of residues 1 to 6 of SEQ ID NO:2; a polypeptide consistingof residues 26 to 54 of SEQ ID NO:2; a polypeptide consisting ofresidues 82 to 94 of SEQ ID NO:2; a polypeptide consisting of residues110 to 117 of SEQ ID NO:2; a polypeptide consisting of residues 110 to127 of SEQ ID NO:2; a polypeptide consisting of residues 122 to 127 ofSEQ ID NO:2; a polypeptide consisting of residues 122 to 136 of SEQ IDNO:2; a polypeptide consisting of residues 131 to 136 of SEQ ID NO:2; apolypeptide consisting of residues 131 to 146 of SEQ ID NO:2; apolypeptide consisting of residues 139 to 146 of SEQ ID NO:2; apolypeptide consisting of residues 154 to 177 of SEQ ID NO:2; apolypeptide consisting of residues 187 to 197 of SEQ ID NO:2; apolypeptide consisting of residues 187 to 207 of SEQ ID NO:2; apolypeptide consisting of residues 202 to 207 of SEQ ID NO:2; apolypeptide consisting of residues 282 to 289 of SEQ ID NO:2; apolypeptide consisting of residues 282 to 301 of SEQ ID NO:2; apolypeptide consisting of residues 295 to 301 of SEQ ID NO:2; apolypeptide consisting of residues 358 to 365 of SEQ ID NO:2; apolypeptide consisting of residues 358 to 397 of SEQ ID NO:2; and apolypeptide consisting of residues 387 to 397 of SEQ ID NO:2; whereinthe polypeptide elicits an immune response in the animal to produce theantibody; and isolating the antibody from the animal. Within anembodiment the antibody which binds to a residues 1 to 397 of SEQ IDNO:2. Within another embodiment, the antibody is a monoclonal antibody.

[0016] Within another aspect the invention provides a method formodulating cell-cell interactions by combining the polypeptide ofresidues 148 to 397, with cells in vivo and in vitro. Within anembodiment the cells are derived from tissues selected from the groupconsisting of: a) tissues from pancreas; b) tissues from colon; c)tissues from small intestine; d) tissues from bladder; e) tissues fromprostate; f) tissues from myometrium; and g) tissues from breast.

[0017] Within another aspect the invention provides a method formodulating glycoprotein and glycolipid biosynthesis by combining thepolypeptide according to claim 1, with cells in vivo and in vitro.Within an embodiment the cells are derived from tissues selected fromthe group consisting of: a) tissues from pancreas; b) tissues fromcolon; c) tissues from small intestine; d) tissues from bladder; e)tissues from prostate; f) tissues from myometrium; and g) tissues frombreast.

[0018] Within another aspect, the invention provides a ethod ofdetecting a molecule which binds to a polypeptide comprising contactingthe polypeptide with a test sample containg the molecule wherein thepolypeptide comprises residues 148 to 397 of SEQ ID NO:2 and whereby themolecule binds the polypeptide.

[0019] These and other aspects of the invention will become evident uponreference to the following detailed description of the invention andattached drawings.

DETAILED DESCRIPTION OF THE INVENTION

[0020] Prior to setting forth the invention in detail, it may be helpfulto the understanding thereof to define the following terms:

[0021] The term “affinity tag” is used herein to denote a polypeptidesegment that can be attached to a second polypeptide to provide forpurification of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-1210,1988), streptavidin binding peptide, maltose binding protein (Guan etal., Gene 67:21-30, 1987), cellulose binding protein, thioredoxin,ubiquitin, T7 polymerase, or other antigenic epitope or binding domain.See, in general, Ford et al., Protein Expression and Purification 2:95-107, 1991. DNAs encoding affinity tags and other reagents areavailable from comrnercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.; New England Biolabs, Beverly, Mass.; Eastman Kodak,New Haven, Conn.).

[0022] The term “allelic variant” is used herein to denote any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

[0023] The terms “amino-terminal” and “carboxyl-terminal” are usedherein to denote positions within polypeptides. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide to denote proximity or relative position. Forexample, a certain sequence positioned carboxyl-terminal to a referencesequence within a polypeptide is located proximal to the carboxylterminus of the reference sequence, but is not necessarily at thecarboxyl terminus of the complete polypeptide.

[0024] The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

[0025] The term “complements of a polynucleotide molecule” is apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence. For example, thesequence 5′ ATGCACGGG 3′ is complementary to 5′CCCGTGCAT 3′.

[0026] The term “contig” denotes a polynucleotide that has a contiguousstretch of identical or complementary sequence to anotherpolynucleotide. Contiguous sequences are said to “overlap” a givenstretch of polynucleotide sequence either in their entirety or along apartial stretch of the polynucleotide. For example, representativecontigs to the polynucleotide sequence 5′-ATGGAGCTT-3′ are5′-AGCTTgagt-3′ and 3′-tcgacTACC-5′.

[0027] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

[0028] The term “expression vector” is used to denote a DNA molecule,linear or circular, that comprises a segment encoding a polypeptide ofinterest operably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

[0029] The term “isolated”, when applied to a polynucleotide, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include CDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78, 1985).

[0030] An “isolated” polypeptide or protein is a polypeptide or proteinthat is found in a condition other than its native environment, such asapart from blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

[0031] “Operably linked” means that two or more entities are joinedtogether such that they function in concert for their intended purposes.When referring to DNA segments, the phrase indicates, for example, thatcoding sequences are joined in the correct reading frame, andtranscription initiates in the promoter and proceeds through the codingsegment(s) to the terminator. When referring to polypeptides, “operablylinked” includes both covalently (e.g., by disulfide bonding) andnon-covalently (e.g., by hydrogen bonding, hydrophobic interactions, orsalt-bridge interactions) linked sequences, wherein the desiredfunction(s) of the sequences are retained.

[0032] The term “ortholog” or “species homolog”, denotes a polypeptideor protein obtained from one species that is the functional counterpartof a polypeptide or protein from a different species. Sequencedifferences among orthologs are the result of speciation.

[0033] “Paralogs” are distinct but structurally related proteins made byan organism. Paralogs are believed to arise through gene duplication.For example, a-globin, b-globin, and myoglobin are paralogs of eachother.

[0034] A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

[0035] A “polypeptide” is a polymer of amino acid residues joined bypeptide bonds, whether produced naturally or synthetically. Polypeptidesof less than about 10 amino acid residues are commonly referred to as“peptides”.

[0036] The term “promoter” is used herein for its art-recognized meaningto denote a portion of a gene containing DNA sequences that provide forthe binding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

[0037] A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

[0038] The term “receptor” denotes a cell-associated protein that bindsto a bioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain or multi-peptide structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. Binding of ligand to receptorresults in a conformational change in the receptor that causes aninteraction between the effector domain and other molecule(s) in thecell. This interaction in turn leads to an alteration in the metabolismof the cell. Metabolic events that are linked to receptor-ligandinteractions include gene transcription, phosphorylation,dephosphorylation, increases in cyclic AMP production, mobilization ofcellular calcium, mobilization of membrane lipids, cell adhesion,hydrolysis of inositol lipids and hydrolysis of phospholipids. Ingeneral, receptors can be membrane bound, cytosolic or nuclear;monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergicreceptor) or multimeric (e.g., PDGF receptor, growth hormone receptor,IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptorand IL-6 receptor).

[0039] The term “secretory signal sequence” denotes a DNA sequence thatencodes a polypeptide (a “secretory peptide”) that, as a component of alarger polypeptide, directs the larger polypeptide through a secretorypathway of a cell in which it is synthesized. The larger polypeptide iscommonly cleaved to remove the secretory peptide during transit throughthe secretory pathway.

[0040] A “segment” is a portion of a larger molecule (e.g.,polynucleotide or polypeptide) having specified attributes. For example,a DNA segment encoding a specified polypeptide is a portion of a longerDNA molecule, such as a plasmid or plasmid fragment, that, when readfrom the 5′ to the 3′ direction, encodes the sequence of amino acids ofthe specified polypeptide.

[0041] The term “splice variant” is used herein to denote alternativeforms of RNA transcribed from a gene. Splice variation arises naturallythrough use of alternative splicing sites within a transcribed RNAmolecule, or less commonly between separately transcribed RNA molecules,and may result in several mRNAs transcribed from the same gene. Splicevariants may encode polypeptides having altered amino acid sequence. Theterm splice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

[0042] Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

[0043] All references cited herein are incorporated by reference intheir entirety.

[0044] The present invention is based upon the discovery of a novel cDNAsequence (SEQ ID NO:1) and corresponding polypeptide (SEQ ID NO:2)having homology to a family of proteins, the β1→3 galactosyltransferases(β1→3GalTases). β1→3GalTases are the β3 subfamily of humangalactosyltransferases (β3Gal-T family) which includes HSY15014(Kolbinger, F. et al., Journal of Biol. Chem. 273: 433-440, 1998),HSGALT3, HSGALT4, (Amado, M. et al., ibid) and E07739 Katsutoshi, S. etal., Japanese patent, JP 1994181759-A/1). β1→3GalTases are responsiblefor transferring galactose to carbohydrate chains during biosynthesis.It has been predicted that β1→3GalTases are in the alpha/beta barrel(TIM barrel) folding class of enzymes, similar to otherglycosyltransferases such as the alpha-amylases and beta-glycanases(Yuan, Y. et al., Cell 88:9-11, 1997). Also in the β3Gal-T family is theDrosophila melanogaster Brainiac, (BRN) (Goode, S. et al., Devel. Biol.178:35-50, 1996), known as “putative neurogenic secreted signalingprotein” or NSSP. BRN is required for epithelial development. Thisactivity may be due to possible cell interactions between the membranebound glycosyltransferase and oligosaccharide substrates on adjacentcell surfaces (Shur, ibid). Thus, β3Gal-T family members are also knownas neurogenic secreted signal peptides. See, for example, Shur, B. D.,ibid, and Amado, M. et al., ibid. This novel polypeptide and itspolynucleotides have been designated znssp2.

[0045] The β3Gal-Ts are predicted to be Type II transmembrane proteins.An ortholog to E07739, is AF029790 (Hennet, T. et al., Journal of Biol.Chem. 273:58-65, 1998), which is claimed to be a Type II transmembranedomain based on hydrophobicity analysis. However, due to the closeproximity of this domain to the initiating methionine and lack ofpositively charged residues preceding the domain it is possible thatAF029790 is not membrane bound but rather a extracellular secretedprotein.

[0046] The novel znssp2 polypeptide-encoding polynucleotides of thepresent invention were initially identified by searching an EST databasefor open reading frames with similarity to BRN. The insert of anexpressed sequence tag corresponding to nucleotides 673 to 1532 of SEQID NO:1 was used to obtain a clone that had been isolated from a bonemarrow library. Analysis of the DNA encoding a znssp2 polypeptide (SEQID NO: 1) revealed an open reading frame encoding 397 amino acids (SEQID NO: 2). Znssp2 shares homology with β3Gal-T's which are predicted tobe Type II membrane proteins. Znssp2 shows the highest similarity toHSGALT3, at 34% amino acid identity over the region of amino acids fromresidue 148 to residue 348 of SEQ ID NO:2. Amino acid residues 19 to 147of SEQ ID NO:2 are predicted to form a “stem” domain, and amino acidresidues 148 to 397 of SEQ ID NO:2 are predicted to form a “catalytic”domain.

[0047] Due to the close proximity of the hydrophobic domain (residues 1to 18 of SEQ ID NO:2) to the initiation methionine, and the lack ofpositively charged residues preceding this domain, it is possible thatznssp2 is a secreted protein comprising a signal peptide of 18 aminoacid residues (residues 1-18 of SEQ ID NO:2) and a mature polypeptide of379 amino acids (residues 19 to 397 of SEQ ID NO:2). Conservednegatively charged amino acid residues 202, 208, 216, 248, 333, and 334of SEQ ID NO:2 are contained within the catalytic domain. Additionally,the sequence of amino acid residues from residue 333 to 338 isrepresentative of a peptide motif of this family. This motif is furtherdescribed by the following amino acid residue profile: [D,E] [D] [V][F,Y] [L,T,V] [G]. Those skilled in the art will recognize thatpredicted domain boundaries are approximations based on primary sequencecontent, and may vary slightly; however, such estimates are generallyaccurate to within ±5 amino acid residues.

[0048] The present invention also provides post translationally modifiedpolypeptides or polypeptide fragments. A potential N-linkedglycosylation site can be found at amino acid residue 220 of SEQ IDNO:2. Post translational modifications in members of the β3Gal-T familymay regulate whether the protein is expressed in the Golgi or on thesurface of the cell. Other examples of post translational modificationsinclude proteolytic cleavage, disulfide bonding and hydroxylation.

[0049] Additionally, znssp2 has 29% homology to the BRN gene.

[0050] The present invention also includes the murine ortholog of znssp2(znssp2-m) which was identified in a mouse EST database. Thepolynucleotide, polypeptide, and degenerate sequences of znssp2-m areshown in SEQ ID NOs:12, 13, and 14, respectively.

[0051] Analysis of the tissue distribution of znssp2 was performed bythe Northern blotting technique using Human Multiple Tissue and MasterDot Blots. A very strong signal of 1.2 kb was seen in pancreas. A strongsignal was seen in colon; with less strong signals seen in spinal cord,bone marrow, small intestine, and peripheral blood leukocytes. Faintersignals were seen in heart, lung, spleen, prostate, stomach, thyroid,trachea, placenta, skeletal muscle, kidney and lymph node.

[0052] The highly conserved, negatively charged residues at positions202, 208, 216, 248, 333, and 334 of SEQ ID NO:2 and the amino acidsequence between 333 and 338 of znssp2 can be used as a tool to identifynew family members. For instance, reverse transcription-polymerase chainreaction (RT-PCR) can be used to amplify sequences encoding the znssp2polynucleotide from RNA obtained from a variety of tissue sources orcell lines. In particular, highly degenerate primers designed from theznssp2 sequences are useful for this purpose. The present inventionfurther provides polynucleotide molecules, including DNA and RNAmolecules, encoding znssp2 proteins. The polynucleotides of the presentinvention include the sense strand; the anti-sense strand; and the DNAas double-stranded, having both the sense and anti-sense strand annealedtogether by their respective hydrogen bonds. Representative DNAsequences encoding znssp2 proteins are set forth in SEQ ID NOs:1, 3, 12and 14. DNA sequences encoding other znssp2 proteins can be readilygenerated by those of ordinary skill in the art based on the geneticcode.

[0053] The present invention also provides polynucleotide molecules,including DNA and RNA molecules, that encode the znssp2 polypeptidesdisclosed herein. Those skilled in the art will readily recognize that,in view of the degeneracy of the genetic code, considerable sequencevariation is possible among these polynucleotide molecules. SEQ ID NO:3is a degenerate DNA sequence that encompasses all DNAs that encode theznssp2 polypeptide of SEQ ID NO:2. SEQ ID NO:14 is a degenerate DNAsequence that encompasses all DNAs that encode the znssp2 polypeptide ofSEQ ID NO:13. Those skilled in the art will recognize that thedegenerate sequence of SEQ ID NO:3 also provides all RNA sequencesencoding SEQ ID NO:2 by substituting U for T and in the same manner, allthe degenerate sequences of SEQ ID NO:14 also provides all RNA sequencesencoding SEQ ID NO:13. Thus, znssp2 polypeptide-encoding polynucleotidescomprising nucleotide 1 to nucleotide 1191 of SEQ ID NO:3 and their RNAequivalents, and the polypeptide-encoding polynucleotides comprisingnucleotide 1 to nucleotide 1167 of SEQ ID NO:14 are contemplated by thepresent invention. Table 1 sets forth the one-letter codes used withinSEQ ID NO:3 to denote degenerate nucleotide positions. “Resolutions” arethe nucleotides denoted by a code letter. “Complement” indicates thecode for the complementary nucleotide(s). For example, the code Ydenotes either C or T, and its complement R denotes A or G, A beingcomplementary to T, and G being complementary to C. TABLE 1 NucleotideResolution Complement Resolution A A T T C C G G G G C C T T A A R A|G YC|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T W A|T H A|C|TD A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T NA|C|G|T

[0054] The degenerate codons used in SEQ ID NOs:3 and 14, encompassingall 5 possible codons for a given amino acid, are set forth in Table 2.TABLE 2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

[0055] One of ordinary skill in the art will appreciate that someambiguity is introduced in determining a degenerate codon,representative of all possible codons encoding each amino acid. Forexample, the degenerate codon for serine (WSN) can, in somecircumstances, encode arginine (AGR), and the degenerate codon forarginine (MGN) can, in some circumstances, encode serine (AGY). Asimilar relationship exists between codons encoding phenylalanine andleucine. Thus, some polynucleotides encompassed by the degeneratesequence may encode variant amino acid sequences, but one of ordinaryskill in the art can easily identify such variant sequences by referenceto the amino acid sequence of SEQ ID NOs:2 or 13. Variant sequences canbe readily tested for functionality as described herein.

[0056] One of ordinary skill in the art will also appreciate thatdifferent species can exhibit “preferential codon usage.” In general,see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al.Curr. Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 2). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commronlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequences disclosed in SEQID NOs:3 and 14 serve as a templates for optimizing expression ofpolynucleotides in various cell types and species commonly used in theart and disclosed herein. Sequences containing preferential codons canbe tested and optimized for expression in various species, and testedfor functionality as disclosed herein.

[0057] Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NOs:1or 12, other polynucleotide probes, primers, fragments and sequencesrecited herein or sequences complementary thereto. Polynucleotidehybridization is well known in the art and widely used for manyapplications, see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989;Ausubel et al., eds., Current Protocols in Molecular Biology, John Wileyand Sons, Inc., NY, 1987; Berger and Kimmel, eds., Guide to MolecularCloning Techniques, Methods in Enzymology, volume 152, 1987 and Wetmur,Crit. Rev. Biochem. Mol. Biol. 26:227-59, 1990. Polynucleotidehybridization exploits the ability of single stranded complementarysequences to form a double helix hybrid. Such hybrids include DNA-DNA,RNA-RNA and DNA-RNA.

[0058] Hybridization will occur between sequences which contain somedegree of complementarity. Hybrids can tolerate mismatched base pairs inthe double helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1Cfor every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases. Hybridization buffers generally containblocking agents such as Denhardt's solution (Sigma Chemical Co., St.Louis, Mo.), denatured salmon sperm DNA, milk powders (BLOTTO), heparinor SDS, and a Na⁺ source, such as SSC (1×SSC: 0.15 M NaCl, 15 mM sodiumcitrate) or SSPE (1×SSPE: 1.8 M NaCl, 10 mM NaH₂PO₄, 1 mM EDTA, pH 7.7).By decreasing the ionic concentration of the buffer, the stability ofthe hybrid is increased. Typically, hybridization buffers contain frombetween 10 mM to 1 M Na⁺. Premixed hybridization solutions are alsoavailable from commercial sources such as Clontech Laboratories (PaloAlto, Calif.) and Promega Corporation (Madison, Wis.) for use accordingto manufacturer's instruction. Addition of destabilizing or denaturingagents such as formamide, tetralkylammonium salts, guanidinium cationsor thiocyanate cations to the hybridization solution will alter theT_(m) of a hybrid. Typically, formamide is used at a concentration of upto 50% to allow incubations to be carried out at more convenient andlower temperatures. Formamide also acts to reduce non-specificbackground when using RNA probes.

[0059] Stringent hybridization conditions encompass temperatures ofabout 5-25° C. below the thermal melting point (T_(m)) of the hybrid anda hybridization buffer having up to 1 M Na⁺. Higher degrees ofstringency at lower temperatures can be achieved with the addition offormamide which reduces the T_(m) of the hybrid about 1° C. for each 1%formamide in the buffer solution. Generally, such stringent conditionsinclude temperatures of 20-70° C. and a hybridization buffer containing5× to 6×SSC and 0-50% formamide. A higher degree of stringency can beachieved at temperatures of from 40-70° C. with a hybridization bufferhaving 3× to 4×SSC and from 0-50% formamide. Highly stringent conditionstypically encompass temperatures of 42-70° C. with a hybridizationbuffer having up to 2×SSC and 0-50% formamide. Different degrees ofstringency can be used during hybridization and washing to achievemaximum specific binding to the target sequence. Typically, the washesfollowing hybridization are performed at increasing degrees ofstringency to remove non-hybridized polynucleotide probes fromhybridized complexes.

[0060] The above conditions are meant to serve as a guide and it is wellwithin the abilities of one skilled in the art to adapt these conditionsfor use with a particular polypeptide hybrid. The T_(m) for a specifictarget sequence is the temperature (under defined conditions) at which50% of the target sequence will hybridize to a perfectly matched probesequence. Those conditions that influence the T_(m) include, the sizeand base pair content of the polynucleotide probe, the ionic strength ofthe hybridization solution, and the presence of destabilizing agents inthe hybridization solution.

[0061] Numerous equations for calculating T_(m) are known in the art,see for example (Sambrook et al., ibid.; Ausubel et al., ibid.; Bergerand Kimmel, ibid. and Wetmur, ibid.) and are specific for DNA, RNA andDNA-RNA hybrids and polynucleotide probe sequences of varying length.Sequence analysis software such as Oligo 4.0 and Primer Premier, as wellas sites on the Internet, are available tools for analyzing a givensequence and calculating T_(m) based on user defined criteria. Suchprograms can also analyze a given sequence under defined conditions andsuggest suitable probe sequences. Typically, hybridization of longerpolynucleotide sequences, >50 bp, is done at temperatures of about20-25° C. below the calculated T_(m). For smaller probes, <50 bp,hybridization is typically carried out at the T_(m) or 5-10° C. below.This allows for the maximum rate of hybridization for DNA-DNA andDNA-RNA hybrids.

[0062] As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of znssp2 RNA. Such tissues and cells areidentified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA77:5201, 1980), and include pancreas, colon, spinal cord, smallintestine, heart, lung, spleen, kidney, prostate, peripheral bloodleukocytes, stomach, thyroid, and trachea. Total RNA can be preparedusing guanidine isothiocyante extraction followed by isolation bycentrifugation in a CsCl gradient (Chirgwin et al., Biochemistry18:52-94, 1979). Poly (A)⁺ RNA is prepared from total RNA using themethod of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972).Complementary DNA (cDNA) is prepared from poly(A)⁺ RNA using knownmethods. In the alternative, genomic DNA can be isolated.Polynucleotides encoding znssp2 polypeptides are then identified andisolated by, for example, hybridization or PCR.

[0063] A full-length clone encoding znssp2 can be obtained byconventional cloning procedures. Complementary DNA (cDNA) clones arepreferred, although for some applications (e.g., expression intransgenic animals) it may be preferable to use a genomic clone, or tomodify a cDNA clone to include at least one genomic intron. Methods forpreparing cDNA and genomic clones are well known and within the level ofordinary skill in the art, and include the use of the sequence disclosedherein, or parts thereof, for probing or priming a library. Expressionlibraries can be probed with antibodies to znssp2, or fragments thereof,or other specific binding partners.

[0064] The invention also provides isolated and purified znssp2polynucleotide probes. Such polynucleotide probes can be RNA or DNA. DNAcan be either cDNA or genomic DNA. Polynucleotide probes are single ordouble-stranded DNA or RNA, generally synthetic oligonucleotides, butmay be generated from cloned cDNA or genomic sequences and willgenerally comprise at least 16 nucleotides, more often from 17nucleotides to 25 or more nucleotides, sometimes 40 to 60 nucleotides,and in some instances a substantial portion, domain or even the entireznssp2 gene or cDNA. The synthetic oligonucleotides of the presentinvention have at least 75% identity to a representative znssp2 DNAsequence (SEQ ID NOs:1, 3, 12 or 14) or their complements. The inventionalso provides oligonucleotide probes or primers comprising at least 14contiguous nucleotides of a polynucleotide of SEQ ID NOs: 1, 3, 12, or14 or a sequence complementary to SEQ ID NOs: 1, 3, 12 or 14.

[0065] Regions from which to construct probes include the 5′ and/or 3′coding sequences, substrate binding regions, and signal sequences, andthe like. Techniques for developing polynucleotide probes andhybridization techniques are known in the art, see for example, Ausubelet al., eds., Current Protocols in Molecular Biology, John Wiley andSons, Inc., NY, 1991. For use as probes, the molecules can be labeled toprovide a detectable signal, such as with an enzyme, biotin, aradionuclide, fluorophore, chemiluminescer, paramagnetic particle andthe like, which are commercially available from many sources, such asMolecular Probes, Inc., Eugene, Oreg., and Amersham Corp., ArlingtonHeights, Ill., using techniques that are well known in the art. Suchprobes can also be used in hybridizations to detect the presence orquantify the amount of znssp2 gene or mRNA transcript in a sample.Znssp2 polynucleotide probes could be used to hybridize to DNA or RNAtargets for diagnostic purposes, using such techniques such asfluorescent in situ hybridization (FISH) or immunohistochemistry.Polynucleotide probes can be used to identify genes encoding znssp2-likeproteins. For example, znssp2 polynucleotides can be used as primersand/or templates in PCR reactions to identify other novel members of theUDP-glycosyltransferase family. Such probes can also be used to screenlibraries for related sequences encoding novel UDP-glycosyltransferases.Such screening would be carried out under conditions of low stringencywhich would allow identification of sequences which are substantiallyhomologous, but not requiring complete homology to the probe sequence.Such methods and conditions are well known in the art, see, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor, N.Y., 1989. Such low stringency conditions couldinclude hybridization temperatures less than 42° C., formamideconcentrations of less than 50% and moderate to low concentrations ofsalt. Libraries may be made of genomic DNA or cDNA. Polynucleotideprobes are also useful for Southern, Northern, or dot blots, colony andplaque hybridization and in situ hybridization. Mixtures of differentznssp2 polynucleotide probes can be prepared which would increasesensitivity or the detection of low copy number targets, in screeningsystems.

[0066] The polynucleotides of the present invention can also besynthesized using DNA synthesizers. Currently the method of choice isthe phosphoramidite method. If chemically synthesized double strandedDNA is required for an application such as the synthesis of a gene or agene fragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 bp) is technically straightforwardand can be accomplished by synthesizing the complementary strands andthen annealing them. For the production of longer genes (>300 bp),however, special strategies must be invoked, because the couplingefficiency of each cycle during chemical DNA synthesis is seldom 100%.To overcome this problem, synthetic genes (double-stranded) areassembled in modular form from single-stranded fragments that are from20 to 100 nucleotides in length. See Glick and Pasternak, MolecularBiotechnology, Principles & Applications of Recombinant DNA, (ASM Press,Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-56,1984 and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-7, 1990.

[0067] The present invention further provides counterpart polypeptidesand polynucleotides from other species (orthologs). These speciesinclude, but are not limited to mammalian, avian, amphibian, reptile,fish, insect and other vertebrate and invertebrate species. Ofparticular interest are znssp2 polypeptides from other mammalianspecies, including murine, porcine, ovine, bovine, canine, feline,equine, and other primate polypeptides. Orthologs of human znssp2 can becloned using information and compositions provided by the presentinvention in combination with conventional cloning techniques. Forexample, a cDNA can be cloned using mRNA obtained from a tissue or celltype that expresses znssp2 as disclosed herein. Suitable sources of mRNAcan be identified by probing Northern blots with probes designed fromthe sequences disclosed herein. A library is then prepared from mRNA ofa positive tissue or cell line. A znssp2-encoding cDNA can then beisolated by a variety of methods, such as by probing with a complete orpartial human cDNA or with one or more sets of degenerate probes basedon the disclosed sequences. A cDNA can also be cloned using thepolymerase chain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202),using primers designed from the representative human znssp2 sequencedisclosed herein. Within an additional method, the cDNA library can beused to transform or transfect host cells, and expression of the cDNA ofinterest can be detected with an antibody to znssp2 polypeptide. Similartechniques can also be applied to the isolation of genomic clones.

[0068] Those skilled in the art will recognize that the sequencesdisclosed in SEQ ID NOd:1, and 12 represent single alleles of human andmouse znssp2, respectively and that allelic variation and alternativesplicing are expected to occur. Allelic variants of this sequence can becloned by probing cDNA or genomic libraries from different individualsaccording to standard procedures. Allelic variants of the DNA sequenceshown in SEQ ID NOs:1 and 12, including those containing silentmutations and those in which mutations result in amino acid sequencechanges, are within the scope of the present invention, as are proteinswhich are allelic variants of SEQ ID NOs:2 and 13. cDNAs generated fromalternatively spliced mRNAs, which retain the properties of the znssp2polypeptide are included within the scope of the present invention, asare polypeptides encoded by such cDNAs and mRNAs. Allelic variants andsplice variants of these sequences can be cloned by probing cDNA orgenomic libraries from different individuals or tissues according tostandard procedures known in the art.

[0069] The present invention also provides isolated znssp2 polypeptidesthat are substantially homologous to the polypeptides of SEQ ID NOs:2and 12 and their orthologs. The term “substantially homologous” is usedherein to denote polypeptides having 50%, preferably 60%, morepreferably at least 80%, sequence identity to the sequences shown in SEQID NOs:2 and 13 or their orthologs. Such polypeptides will morepreferably be at least 90% identical, and most preferably 95% or moreidentical to SEQ ID NOs:2 and 13 or their orthologs.) Percent sequenceidentity is determined by conventional methods. See, for example,Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff andHenikoff, Proc. Natl. Acad. Sci. USA 89:10915-9, 1992. Briefly, twoamino acid sequences are aligned to optimize the alignment scores usinga gap opening penalty of 10, a gap extension penalty of 1, and the“blosum 62” scoring matrix of Henikoff and Henikoff (ibid.) as shown inTable 3 (amino acids are indicated by the standard one-letter codes).The percent identity is then calculated as:$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\begin{matrix}\left\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {plus}\quad {the}} \right. \\{{number}\quad {of}\quad {gaps}\quad {introduced}\quad {into}\quad {the}\quad {longer}}\end{matrix} \\\left. {{sequence}\quad {in}\quad {order}\quad {to}\quad {align}\quad {the}\quad {two}\quad {sequences}} \right\rbrack\end{matrix}} \times 100$

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −2 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1 1−2 −1 3 3 −2 2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 2 0 −3 −14

[0070] Sequence identity of polynucleotide molecules is determined bysimilar methods using a ratio as disclosed above.

[0071] Those skilled in the art appreciate that there are manyestablished algorithms available to align two amino acid sequences. The“FASTA” similarity search algorithm of Pearson and Lipman is a suitableprotein alignment method for examining the level of identity shared byan amino acid sequence disclosed herein and the amino acid sequence of aputative variant znssp2. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

[0072] Briefly, FASTA first characterizes sequence similarity byidentifying regions shared by the query sequence (e.g., SEQ ID NO:2) anda test sequence that have either the highest density of identities (ifthe ktup variable is 1) or pairs of identities (if ktup=2), withoutconsidering conservative amino acid substitutions, insertions, ordeletions. The ten regions with the highest density of identities arethen rescored by comparing the similarity of all paired amino acidsusing an amino acid substitution matrix, and the ends of the regions are“trimmed” to include only those residues that contribute to the highestscore. If there are several regions with scores greater than the“cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

[0073] FASTA can also be used to determine the sequence identity ofnucleic acid molecules using a ratio as disclosed above. For nucleotidesequence comparisons, the ktup value can range between one to six,preferably from four to six.

[0074] The present invention includes nucleic acid molecules that encodea polypeptide having one or more conservative amino acid changes,compared with the amino acid sequences of SEQ ID NOs:2 and 13. TheBLOSUM62 table is an amino acid substitution matrix derived from about2,000 local multiple alignments of protein sequence segments,representing highly conserved regions of more than 500 groups of relatedproteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915(1992)). Accordingly, the BLOSUM62 substitution frequencies can be usedto define conservative amino acid substitutions that may be introducedinto the amino acid sequences of the present invention. As used herein,the language “conservative amino acid substitution” refers to asubstitution represented by a BLOSUM62 value of greater than −1. Forexample, an amino acid substitution is conservative if the substitutionis characterized by a BLOSUM62 value of 0, 1, 2, or 3. Conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 1 (e.g., 1, 2 or 3), while more conservative amino acidsubstitutions are characterized by a BLOSUM62 value of at least 2 (e.g.,2 or 3).

[0075] Variant znssp2 polypeptides or substantially homologous znssp2polypeptides are characterized as having one or more amino acidsubstitutions, deletions or additions. These changes are preferably of aminor nature, that is conservative amino acid substitutions (see Table4) and other substitutions that do not significantly affect the foldingor activity of the polypeptide; small deletions, typically of one toabout 30 amino acids; and small amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or an affinity tag. The present inventionthus includes polypeptides of from 397 to 410 amino acid residues thatcomprise a sequence that is at least 70%, preferably at least 80%, andmore preferably 90% or more identical to the corresponding region of SEQID NO:2. Polypeptides comprising affinity tags can further comprise aproteolytic cleavage site between the znssp2 polypeptide and theaffinity tag. Preferred such sites include thrombin cleavage sites andfactor Xa cleavage sites. TABLE 4 Conservative amino acid substitutionsBasic: arginine lysine histidine Acidic: glutamic acid aspartic acidPolar: glutamine asparagine Hydrophobic: leucine isoleucine valineAromatic: phenylalanine tryptophan tyrosme Small: glycine alanine serinethreonine methiomne

[0076] The present invention further provides a variety of otherpolypeptide fusions and related multimeric proteins comprising one ormore polypeptide fusions. For example, a znssp2 polypeptide can beprepared as a fusion to a dimerizing protein as disclosed in U.S. Pat.Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in thisregard include immunoglobulin constant region domains.Immunoglobulin-znssp2 polypeptide fusions can be expressed ingenetically engineered cells to produce a variety of multimeric znssp2analogs. Auxiliary domains can be fused to znssp2 polypeptides to targetthem to specific cells, tissues, or macromolecules (e.g., pancreas,colon, spinal cord, bone marrow, heart, and small intestine etc.). Forexample, a protease, or ablation antibody polypeptide or protein couldbe targeted to a predetermined cell type by fusing a said protease, orablation antibody polypeptide to a ligand that specifically binds to areceptor or receptor-like complementary molecule on the surface of thetarget cell, such as, pancreas, colon, spinal cord, or bone marrow. Inthis way, polypeptides and proteins can be targeted for therapeutic ordiagnostic purposes. Such beta-1,3-galactosyltransferase polypeptidescan be fused to two or more moieties, such as an affinity tag forpurification and a targeting domain. Polypeptide fusions can alsocomprise one or more cleavage sites, particularly between domains. See,Tuan et al., Connective Tissue Research 34:1-9, 1996.

[0077] Polypeptide fusions of the present invention will generallycontain not more than about 1,700 amino acid residues, not more thanabout 1,200 residues, or not more than about 1,000 residues, and will inmany cases be considerably smaller. For example, residues of znssp2polypeptide can be fused to E. coli β-galactosidase (1,021 residues; seeCasadaban et al., J. Bacteriol. 143:971-980, 1980), a 10-residue spacer,and a 4-residue factor Xa cleavage site. In a second example, residuesof znssp2 polypeptide can be fused to maltose binding protein(approximately 370 residues), a 4-residue cleavage site, and a 6-residuepolyhistidine tag.

[0078] Some proteins in the β3Gal-T family been shown to be expressedintracellulary and are involved in intracellular glycoprotein andglycolipid processing. Other members of this family have been shown tobe extracellularly expressed and are involved in glycoprotein andglycolipid processing (such as in the case of the Tn antigen). Othermembers of the family are expressed extracellularly and are involved incell-cell interactions and intracellular signaling. Thus, molecules ofthe present invention can function as an enzyme both intracellularly andextracellulary, in which case its anti-complementary molecule is asubstrate. Additionally, molecules of the present invention can functionextracellularly and modulate cell-cell interactions. The extracellularbinding of znssp2 to its anti-complementary molecule can cause acellular event in the cell that is expressing it (i.e. znssp2 acts as areceptor or receptor-like molecule), or in the cell expressing theanti-complementary molecule to which it binds (i.e., znssp2 acts as aligand). Additionally, znssp2 can function extracellularly as a solubleenzyme, ligand, receptor or receptor like molecule. Similarly, as anextracellulary expressed znssp2 enzyme, the processing of itsanti-complementary substrate can result in a cellular response (similarto intracellular signaling) in the cell expressing the substrate. Alsoas an extracellularly expressed protein, znssp2 can function to form a“bridge” between cells maintaining their proximity to each other. Thus,for the purposes of this application, znssp2 is referred to as acomplementary molecule and its cognate binding partner is referred to asan anti-complementary molecule.

[0079] The invention also provides soluble znssp2 polypeptides, used toform fusion or chimeric proteins with human Ig, as His-tagged proteins,or FLAG™-tagged proteins. One such construct is comprises residues 19 to397 of SEQ ID NO:2, fused to human Ig. znssp2 or znssp2-Ig chimericproteins are used, for example, to identify the znssp2anti-complementary molecule, including the natural anti-complementarymolecule, as well as agonists and antagonists of the naturalanti-complementary molecule. Using labeled soluble znssp2, cellsexpressing the anti-complementary molecule are identified byfluorescence immunocytometry or immunohistochemistry. The soluble fusionproteins or soluble Ig fusion protein is useful in studying thedistribution of the anti-complementary molecule on tissues or specificcell lineages, and to provide insight into complementarymolecule-anti-complementary molecule biology.

[0080] In an alternative approach, a soluble znssp2 extracellularanti-complementary molecule-binding region can be expressed as a chimerawith immunoglobulin heavy chain constant regions, typically an Fcfragment, which contains two constant region domains and a hinge region,but lacks the variable region. Such fusions are typically secreted asmultimeric molecules, wherein the Fc portions are disulfide bonded toeach other and two enzyme polypeptides are arrayed in close proximity toeach other. Fusions of this type can be used to affinity purify thecognate substrate from solution, as an in vitro assay tool, to blocksignals in vitro by specifically titrating out anti-complementarymolecule, and as antagonists in vivo by administering them to blockanti-complementary molecule stimulation. To purify anti-complementarymolecule, a znssp2-Ig fusion protein (chimera) is added to a samplecontaining the anti-complementary molecule under conditions thatfacilitate complementary moleucle-anti-complementary molecule binding(typically near-physiological temperature, pH, and ionic strength). Thechimera-substrate complex is then separated by the mixture using proteinA, which is immobilized on a solid support (e.g., insoluble resinbeads). The anti-complementary molecule is then eluted usingconventional chemical techniques, such as with a salt or pH gradient. Inthe alternative, the chimera itself can be bound to a solid support,with binding and elution carried out as above. For use in assays, thechimeras are bound to a support via the Fc region and used in an ELISAformat.

[0081] The present invention also includes “functional fragments” ofznssp2 polypeptides and nucleic acid molecules encoding such functionalfragments. Routine deletion analyses of nucleic acid molecules can beperformed to obtain functional fragments of a nucleic acid molecule thatencodes an znssp2 polypeptide. As an illustration, DNA molecules havingthe nucleotide sequences of SEQ ID NOs:1 and 12 can be digested withBal31 nuclease to obtain a series of nested deletions. The fragments arethen inserted into expression vectors in proper reading frame, and theexpressed polypeptides are isolated and tested for cell-cellinteractions, or for the ability to bind anti-znssp2 antibodies. Onealternative to exonuclease digestion is to use oligonucleotide-directedmutagenesis to introduce deletions or stop codons to specify productionof a desired fragment. Alternatively, particular fragments of an znssp2gene can be synthesized using the polymerase chain reaction.

[0082] Standard methods for identifying functional domains arewell-known to those of skill in the art. For example, studies on thetruncation at either or both termini of interferons have been summarizedby Horisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993),Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987), Herschman, “The EGFEnzyme,” in Control of Animal Cell Proliferation, Vol. 1, Boynton etal., (eds.) pages 169-199 (Academic Press 1985), Coumailleau et al., J.Biol. Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995), and Meiselet al., Plant Molec. Biol. 30:1 (1996).

[0083] The present invention also contemplates functional fragments ofan znssp2 gene that has amino acid changes, compared with the amino acidsequences of SEQ ID NOs:2 and 13. A variant znssp2 gene can beidentified on the basis of structure by determining the level ofidentity with nucleotide and amino acid sequences of SEQ ID NOs:1, 2, 12and 13, as discussed above. An alternative approach to identifying avariant gene on the basis of structure is to determine whether a nucleicacid molecule encoding a potential variant znssp2 gene can hybridize toa nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1,as discussed above.

[0084] The proteins of the present invention can also comprisenon-naturally occurring amino acid residues. Non-naturally occurringamino acids include, without limitation, trans-3-methylproline,2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline,N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, teri-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is carried outin a cell-free system comprising an E. coli S30 extract and commerciallyavailable enzymes and other reagents. Proteins are purified bychromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung etal., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci.USA 90:10145-9, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-8, 1996). Within a third method, E. coli cells are cultured inthe absence of a natural amino acid that is to be replaced (e.g.,phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

[0085] A limited number of non-conservative amino acids, amino acidsthat are not encoded by the genetic code, non-naturally occurring aminoacids, and unnatural amino acids may be substituted for znssp2 aminoacid residues.

[0086] Essential amino acids in the polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc.Natl. Acad. Sci. USA 88:4498-502, 1991). In the latter technique, singlealanine mutations are introduced at every residue in the molecule, andthe resultant mutant molecules are tested for biological activity asdisclosed below to identify amino acid residues that are critical to theactivity of the molecule. See also, Hilton et al., J. Biol. Chem.271:4699-708, 1996. Sites of galactosyltransferase activity andcell-cell interactions can also be determined by physical analysis ofstructure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., Science 255:306-12, 1992; Smithet al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett.309:59-64, 1992. The identities of essential amino acids can also beinferred from analysis of homologies with related galactosyltransferasemolecules.

[0087] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner etal., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Neret al., DNA 7:127, 1988).

[0088] Variants of the disclosed znssp2 DNA and polypeptide sequencescan be generated through DNA shuffling as disclosed by Stemmer, Nature370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated byin vitro homologous recombination by random fragmentation of a parentDNA followed by reassembly using PCR, resulting in randomly introducedpoint mutations. This technique can be modified by using a family ofparent DNAs, such as allelic variants or DNAs from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

[0089] Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode active polypeptides (e.g., galactosyltransferaseactivity as evidenced by glycoprotein synthesis, or cell-cellinteractions, such as intracellular signaling) can be recovered from thehost cells and rapidly sequenced using modem equipment. These methodsallow the rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

[0090] Regardless of the particular nucleotide sequence of a variantznssp2 gene, the gene encodes a polypeptide that is characterized by itsanti-complementary molecule binding activity, or by the ability to bindspecifically to an anti-znssp2 antibody. More specifically, variantznssp2 genes encode polypeptides which exhibit greater than 75, 80, or90%, of the activity of polypeptide encoded by the human znssp2 genedescribed herein.

[0091] Using the methods discussed herein, one of ordinary skill in theart can identify and/or prepare a variety of polypeptide fragments orvariants of SEQ ID NO:2 or that retain the galactsyltransferaseproperties, or cell-cell interactions of the wild-type znssp2 protein.Such polypeptides may include additional amino acids from, for example,an extracellular ligand-binding domain of another member of thegalactosyltransferase family as well as part or all of the transmembraneand intracellular domains. Additionally fragments of znssp2 may includeadditional amino acids from the catalytic site of thegalactosyltransferase domains of other family members. Additional aminoacids from affinity tags and the like may also be included.

[0092] For any znssp2 polypeptide, including variants and fusionproteins, one of ordinary skill in the art can readily generate a fullydegenerate polynucleotide sequence encoding that variant using theinformation set forth in Tables 1 and 2 above. Moreover, those of skillin the art can use standard software to devise znssp2 variants basedupon the nucleotide and amino acid sequences described herein.Accordingly, the present invention includes a computer-readable mediumencoded with a data structure that provides at least one of thefollowing sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:12, SEQ ID NO:13, and SEQ ID NO:14. Suitable forms ofcomputer-readable media include magnetic media and optically-readablemedia. Examples of magnetic media include a hard or fixed drive, arandom access memory (RAM) chip, a floppy disk, digital linear tape(DLT), a disk cache, and a ZIP disk. Optically readable media areexemplified by compact discs (e.g., CD-read only memory (ROM),CD-rewritable (RW), and CD-recordable), and digital versatile/videodiscs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).

[0093] The znssp2 polypeptides of the present invention, includingfull-length polypeptides, biologically active fragments, and fusionpolypeptides, can be produced in genetically engineered host cellsaccording to conventional techniques. Suitable host cells are those celltypes that can be transformed or transfected with exogenous DNA andgrown in culture, and include bacteria, fungal cells, and culturedhigher eukaryotic cells. Eukaryotic cells, particularly cultured cellsof multicellular organisms, are preferred. Techniques for manipulatingcloned DNA molecules and introducing exogenous DNA into a variety ofhost cells are disclosed by Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, and Ausubel et al., eds., Current Protocolsin Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

[0094] In general, a DNA sequence encoding a znssp2 polypeptide isoperably linked to other genetic elements required for its expression,generally including a transcription promoter and terminator, within anexpression vector. The vector will also commonly contain one or moreselectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers may be provided on separate vectors, and replicationof the exogenous DNA may be provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers, vectorsand other elements is a matter of routine design within the level ofordinary skill in the art. Many such elements are described in theliterature and are available through commercial suppliers.

[0095] To direct a znssp2 polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of znssp2, or may be derivedfrom another secreted protein (e.g., t-PA) or synthesized de novo. Thesecretory signal sequence is operably linked to the znssp2 DNA sequence,i.e., the two sequences are joined in the correct reading frame andpositioned to direct the newly synthesized polypeptide into thesecretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain secretory signal sequences may be positionedelsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S.Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

[0096] The native secretory signal sequence of the polypeptides of thepresent invention is used to direct other polypeptides into thesecretory pathway. The present invention provides for such fusionpolypeptides. A signal fusion polypeptide can be made wherein asecretory signal sequence derived from a znssp2 polypeptide is beoperably linked to another polypeptide using methods known in the artand disclosed herein. The secretory signal sequence contained in thefusion polypeptides of the present invention is preferably fusedamino-terminally to an additional peptide to direct the additionalpeptide into the secretory pathway. Such constructs have numerousapplications known in the art. For example, these novel secretory signalsequence fusion constructs can direct the secretion of an activecomponent of a normally non-secreted protein, such as a receptor. Suchfusions may be used in vivo or in vitro to direct peptides through thesecretory pathway.

[0097] Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-5, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993, and viral vectors (Miller and Rosman, Bio Techniques 7:980-90,1989; Wang and Finer, Nature Med. 2:714-6, 1996). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Manassas, Va. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

[0098] Drug selection is generally used to select for cultured mammaliancells into which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

[0099] Other higher eukaryotic cells can also be used as hosts,including plant cells, insect cells and avian cells. The use ofAgrobacterium rhizogenes as a vector for expressing genes in plant cellshas been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58,1987. Transformation of insect cells and production of foreignpolypeptides therein is disclosed by Guarino et al., U.S. Pat. No.5,162,222 and WIPO publication WO 94/06463. Insect cells can be infectedwith recombinant baculovirus, commonly derived from Autographacalifornica nuclear polyhedrosis virus (AcNPV). See, King, L. A. andPossee, R. D., The Baculovirus Expression System: A Laboratory Guide,London, Chapman & Hall; O'Reilly, D. R. et al., Baculovirus ExpressionVectors: A Laboratory Manual, New York, Oxford University Press., 1994;and, Richardson, C. D., Ed., Baculovirus Expression Protocols. Methodsin Molecular Biology, Totowa, N.J., Humana Press, 1995. A second methodof making recombinant znssp2 baculovirus utilizes a transposon-basedsystem described by Luckow (Luckow, V. A, et al., J. Virol. 67:4566-79,1993). This system, which utilizes transfer vectors, is sold in theBac-to-Bac™ kit (Life Technologies, Rockville, Md.).

[0100] This system utilizes a transfer vector, pFastBac1™ (LifeTechnologies) containing a Tn7 transposon to move the DNA encoding theznssp2 polypeptide into a baculovirus genome maintained in E. coli as alarge plasmid called a “bacmid.” The pFastBac1™ transfer vector utilizesthe AcNPV polyhedrin promoter to drive the expression of the gene ofinterest, in this case znssp2. However, pFastBac1™ can be modified to aconsiderable degree. The polyhedrin promoter can be removed andsubstituted with the baculovirus basic protein promoter (also known asPcor, p6.9 or MP promoter) which is expressed earlier in the baculovirusinfection, and has been shown to be advantageous for expressing secretedproteins. See, Hill-Perkins, M. S. and Possee, R. D., J. Gen. Virol.71:971-6, 1990; Bonning, B. C. et al., J. Gen. Virol. 75:1551-6, 1994;and, Chazenbalk, G. D., and Rapoport, B., J. Biol. Chem. 270:1543-9,1995. In such transfer vector constructs, a short or long version of thebasic protein promoter can be used. Moreover, transfer vectors can beconstructed which replace the native znssp2 secretory signal sequenceswith secretory signal sequencesz derived from insect proteins. Forexample, a secretory signal sequence from EcdysteroidGlucosyltransferase (EGT), honey bee Melittin (Invitrogen, Carlsbad,Calif.), or baculovirus gp67 (PharMingen, San Diego, Calif.) can be usedin constructs to replace the native znssp2 secretory signal sequence. Inaddition, transfer vectors can include an in-frame fusion with DNAencoding an epitope tag at the C- or N-terminus of the expressed znssp2polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T. etal., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Using a technique known inthe art, a transfer vector containing znssp2 is transformed into E.Coli, and screened for bacmids which contain an interrupted lacZ geneindicative of recombinant baculovirus. The bacmid DNA containing therecombinant baculovirus genome is isolated, using common techniques, andused to transfect Spodoptera frugiperda cells, e.g. Sf9 cells.Recombinant virus that expresses znssp2 is subsequently produced.Recombinant viral stocks are made by methods commonly used the art.

[0101] The recombinant virus is used to infect host cells, typically acell line derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells. Thecells are grown up from an inoculation density of approximately 2-5×10⁵cells to a density of 1-2×10⁶ cells at which time a recombinant viralstock is added at a multiplicity of infection (MOI) of 0.1 to 10, moretypically near 3. Procedures used are generally described in availablelaboratory manuals (King, L. A. and Possee, R. D., ibid.; O'Reilly, D.R. et al., ibid.; Richardson, C. D., ibid.). Subsequent purification ofthe znssp2 polypeptide from the supernatant can be achieved usingmethods described herein.

[0102] Fungal cells, including yeast cells, can also be used within thepresent invention. Yeast species of particular interest in this regardinclude Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Methods for transforming S. cerevisiae cells with exogenousDNA and producing recombinant polypeptides therefrom are disclosed by,for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S.Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S.Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075.Transformed cells are selected by phenotype determined by the selectablemarker, commonly drug resistance or the ability to grow in the absenceof a particular nutrient (e.g., leucine). A preferred vector system foruse in Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be 30 utilized according to the methods ofMcKnight et al., U.S. Pat. No. 4,935,349. Methods for transformingAcremonium chrysogenum are disclosed by Sumino et al., U.S. Pat. No.5,162,228. Methods for transforming Neurospora are disclosed byLambowitz, U.S. Pat. No. 4,486,533.

[0103] The use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed in WIPO Publications WO 97/17450, WO97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use intransforming P. methanolica will commonly be prepared asdouble-stranded, circular plasmids, which are preferably linearizedprior to transformation. For polypeptide production in P. methanolica,it is preferred that the promoter and terminator in the plasmid be thatof a P. methanolica gene, such as a P. methanolica alcohol utilizationgene (AUG1 or AUG2). Other useful promoters include those of thedihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), andcatalase (CAT) genes. To facilitate integration of the DNA into the hostchromosome, it is preferred to have the entire expression segment of theplasmid flanked at both ends by host DNA sequences. A preferredselectable marker for use in Pichia methanolica is a P. methanolica ADE2gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC;EC 4.1.1.21), which allows ade2 host cells to grow in the absence ofadenine. For large-scale, industrial processes where it is desirable tominimize the use of methanol, it is preferred to use host cells in whichboth methanol utilization genes (AUG1 and AUG2) are deleted. Forproduction of secreted proteins, host cells deficient in vacuolarprotease genes (PEP4 and PRB1) are preferred. Electroporation is used tofacilitate the introduction of a plasmid containing DNA encoding apolypeptide of interest into P. methanolica cells. It is preferred totransform P. methanolica cells by electroporation using an exponentiallydecaying, pulsed electric field having a field strength of from 2.5 to4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from1 to 40 milliseconds, most preferably about 20 milliseconds.

[0104] Prokaryotic host cells, including strains of the bacteriaEscherichia coli, Bacillus and other genera are also useful host cellswithin the present invention. Techniques for transforming these hostsand expressing foreign DNA sequences cloned therein are well known inthe art (see, e.g., Sambrook et al., ibid.). When expressing a znssp2polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

[0105] Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

[0106] It is preferred to purify the polypeptides of the presentinvention to ≧80% purity, more preferably to ≧90% purity, even morepreferably ≧95% purity, and particularly preferred is a pharmaceuticallypure state, that is greater than 99.9% pure with respect tocontaminating macromolecules, particularly other proteins and nucleicacids, and free of infectious and pyrogenic agents. Preferably, apurified polypeptide is substantially free of other polypeptides,particularly other polypeptides of animal origin.

[0107] Expressed recombinant znssp2 polypeptides (or chimeric znssp2polypeptides) can be purified using fractionation and/or conventionalpurification methods and media. Ammonium sulfate precipitation and acidor chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitablechromatographic media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred. Exemplary chromatographic media include thosemedia derivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties. Examples of coupling chemistriesinclude cyanogen bromide activation, N-hydroxysuccinimide activation,epoxide activation, sulfhydryl activation, hydrazide activation, andcarboxyl and amino derivatives for carbodiimide coupling chemistries.These and other solid media are well known and widely used in the art,and are available from commercial suppliers. Methods for bindingreceptor and receptor-like complementary polypeptides to support mediaare well known in the art. Selection of a particular method is a matterof routine design and is determined in part by the properties of thechosen support. See, for example, Affinity Chromatography: Principles &Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

[0108] The polypeptides of the present invention can be isolated by acombination of procedures including, but not limited to, anion andcation exchange chromatography, size exclusion, and affinitychromatography. For example, immobilized metal ion adsorption (IMAC)chromatography can be used to purify histidine-rich proteins, includingthose comprising polyhistidine tags. Briefly, a gel is first chargedwith divalent metal ions to form a chelate (Sulkowski, Trends inBiochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to thismatrix with differing affinities, depending upon the metal ion used, andwill be eluted by competitive elution, lowering the pH, or use of strongchelating agents. Other methods of purification include purification ofglycosylated proteins by lectin affinity chromatography and ion exchangechromatography (Methods in Enzymol., Vol. 182, “Guide to ProteinPurification”, M. Deutscher, (ed.), Acad. Press, San Diego, 1990,pp.529-39). Within additional embodiments of the invention, a fusion ofthe polypeptide of interest and an affinity tag (e.g., maltose-bindingprotein, an immunoglobulin domain) may be constructed to facilitatepurification.

[0109] To direct the export of a receptor polypeptide from the hostcell, the receptor DNA is linked to a second DNA segment encoding asecretory peptide, such as a t-PA secretory peptide or a znssp2secretory peptide. To facilitate purification of the secreted receptorpolypeptide, a C-terminal extension, such as a poly-histidine tag,substance P, Flag peptide (Hopp et al., Bio/Technology 6:1204-1210,1988; available from Eastman Kodak Co., New Haven, Conn.) or anotherpolypeptide or protein for which an antibody or other specific bindingagent is available, can be fused to the receptor polypeptide.

[0110] Moreover, using methods described in the art, polypeptidefusions, or hybrid znssp2 proteins, are constructed using regions ordomains of the inventive znssp2 in combination with those of other humangalactosyltransferase family proteins (e.g. HSGALT3, HSGALT4, P3Gal-T2,and p3Gal-T3, or the human species ortholog of Brainiac), orheterologous proteins (Sambrook et al., ibid., Altschul et al., ibid.,Picard, Cur. Opin. Biology, 5:511-5, 1994, and references therein).These methods allow the determination of the biological importance oflarger domains or regions in a polypeptide of interest. Such hybrids mayalter reaction kinetics, binding, constrict or expand the substratespecificity, or alter tissue and cellular localization of a polypeptide,and can be applied to polypeptides of unknown structure.

[0111] Fusion proteins can be prepared by methods known to those skilledin the art by preparing each component of the fusion protein andchemically conjugating them. Alternatively, a polynucleotide encodingboth components of the fusion protein in the proper reading frame can begenerated using known techniques and expressed by the methods describedherein. For example, part or all of a domain(s) conferring a biologicalfunction may be swapped between znssp2 of the present invention with thefunctionally equivalent domain(s) from another family member, such asthe human species ortholog of Brainiac, or other galactosyltransferases,etc. Such domains include, but are not limited to, the hydrophobicregion thought to be a putative secretory signal sequence ortransmembrane domain (residues 1 to 18 of SEQ ID NO:2), and otherconserved motifs such as the β1→3 GalTase homology region (residues 148to 397 of SEQ ID NO:2), and significant domains or regions in thisfamily. Such fusion proteins would be expected to have a biologicalfunctional profile that is the same or similar to polypeptides of thepresent invention or other known galactosyltransferase family proteins(e.g. HSGALT3, HSGALT4, and Brainiac), depending on the fusionconstructed. Moreover, such fusion proteins may exhibit other propertiesas disclosed herein.

[0112] Znssp2 polypeptides or fragments thereof may also beprepared-through chemical synthesis. Znssp2 polypeptides may be monomersor multimers; glycosylated or non-glycosylated; pegylated ornon-pegylated; and may or may not include an initial methionine aminoacid residue.

[0113] Znssp2 polypeptides of the present invention can also besynthesized by exclusive solid phase synthesis, partial solid phasemethods, fragment condensation or classical solution synthesis. Thepolypeptides are preferably prepared by solid phase peptide synthesis,for example as described by Merrifield, J. Am. Chem. Soc. 85:2149, 1963.The synthesis is carried out with amino acids that are protected at thealpha-amino terminus. Trifunctional amino acids with labile side-chainsare also protected with suitable groups to prevent undesired chemicalreactions from occurring during the assembly of the polypeptides. Thealpha-amino protecting group is selectively removed to allow subsequentreaction to take place at the amino-terminus. The conditions for theremoval of the alpha-amino protecting group do not remove the side-chainprotecting groups.

[0114] The alpha-amino protecting groups are those known to be useful inthe art of stepwise polypeptide synthesis. Included are acyl typeprotecting groups (e.g., formyl, trifluoroacetyl, acetyl), aryl typeprotecting groups (e.g., biotinyl), aromatic urethane type protectinggroups [e.g., benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl and9-fluorenylmethyloxy-carbonyl (Fmoc)], aliphatic urethane protectinggroups [e.g., t-butyloxycarbonyl (tBoc), isopropyloxycarbonyl,cyclohexloxycarbonyl] and alkyl type protecting groups (e.g., benzyl,triphenylmethyl). The preferred protecting groups are tboc and Fmoc,thus the peptides are said to be synthesized by tboc and Fmoc chemistry,respectively.

[0115] The side-chain protecting groups selected must remain intactduring coupling and not be removed during the deprotection of theamino-terminus protecting group or during coupling conditions. Theside-chain protecting groups must also be removable upon the completionof synthesis using reaction conditions that will not alter the finishedpolypeptide. In tBoc chemistry, the side-chain protecting groups fortrifunctional amino acids are mostly benzyl based. In Fmoc chemistry,they are mostly tert-butyl or trityl based.

[0116] In tBoc chemistry, the preferred side-chain protecting groups aretosyl for arginine, cyclohexyl for aspartic acid, 4-methylbenzyl (andacetamidomethyl) for cysteine, benzyl for glutamic acid, serine andthreonine, benzyloxymethyl (and dinitrophenyl) for histidine,2-Cl-benzyloxycarbonyl for lysine, formyl for tryptophan and2-bromobenzyl for tyrosine. In Fmoc chemistry, the preferred side-chainprotecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) or2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine,trityl for asparagine, cysteine, glutamine and histidine, tert-butyl foraspartic acid, glutamic acid, serine, threonine and tyrosine, tBoc forlysine and tryptophan.

[0117] For the synthesis of phosphopeptides, either direct orpost-assembly incorporation of the phosphate group is used. In thedirect incorporation strategy, the phosphate group on serine, threonineor tyrosine may be protected by methyl, benzyl, or tert-butyl in Fmocchemistry or by methyl, benzyl or phenyl in tBoc chemistry. Directincorporation of phosphotyrosine without phosphate protection can alsobe used in Fmoc chemistry. In the post-assembly incorporation strategy,the unprotected hydroxyl groups of serine, threonine or tyrosine arederivatized on solid phase with di-tert-butyl-, dibenzyl- ordimethyl-N,N′-diisopropylphosphoramidite and then oxidized bytert-butylhydroperoxide.

[0118] Solid phase synthesis is usually carried out from thecarboxyl-terminus by coupling the alpha-amino protected (side-chainprotected) amino acid to a suitable solid support. An ester linkage isformed when the attachment is made to a chloromethyl, chlortrityl orhydroxymethyl resin, and the resulting polypeptide will have a freecarboxyl group at the C-terminus. Alternatively, when an amide resinsuch as benzhydrylamine or p-methylbenzhydrylamine resin (for tBocchemistry) and Rink amide or PAL resin (for Fmoc chemistry) are used, anamide bond is formed and the resulting polypeptide will have acarboxamide group at the C-terminus. These resins, whether polystyrene-or polyamide-based or polyethyleneglycol-grafted, with or without ahandle or linker, with or without the first amino acid attached, arecommercially available, and their preparations have been described byStewart et al., “Solid Phase Peptide Synthesis” (2nd Edition), (PierceChemical Co., Rockford, Ill., 1984) and Bayer & Rapp Chem. Pept. Prot.3:3 (1986); and Atherton et al., Solid Phase Peptide Synthesis: APractical Approach, IRL Press, Oxford, 1989.

[0119] The C-terminal amino acid, protected at the side chain ifnecessary, and at the alpha-amino group, is attached to a hydroxylmethylresin using various activating agents including dicyclohexylcarbodiimide(DCC), N,N′-diisopropylcarbodiimide (DIPCDI) and carbonyldiimidazole(CDI). It can be attached to chloromethyl or chlorotrityl resin directlyin its cesium tetramethylammonium salt form or in the presence oftriethylamine (TEA) or diisopropylethylamine (DIEA). First amino acidattachment to an amide resin is the same as amide bond formation duringcoupling reactions.

[0120] Following the attachment to the resin support, the alpha-aminoprotecting group is removed using various reagents depending on theprotecting chemistry (e.g., tBoc, Fmoc). The extent of Fmoc removal canbe monitored at 300-320 nim or by a conductivity cell. After removal ofthe alpha-amino protecting group, the remaining protected amino acidsare coupled stepwise in the required order to obtain the desiredsequence.

[0121] Various activating agents can be used for the coupling reactionsincluding DCC, DIPCDI, 2-chloro-1,3-dimethylimidium hexafluorophosphate(CIP), benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluoro-phosphate (BOP) and its pyrrolidine analog (PyBOP),bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP),O-(benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate(HBTU) and its tetrafluoroborate analog (TBTU) or its pyrrolidine analog(HBPyU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumhexafluorophosphate (HATU) and its tetrafluoroborate analog (TATU) orits pyrrolidine analog (HAPyU). The most common catalytic additives usedin coupling reactions include 4-dimethylaminopyridine (DMAP),3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt),N-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt).Each protected amino acid is used in excess (>2.0 equivalents), and thecouplings are usually carried out in N-methylpyrrolidone (NMP) or inDMF, CH2Cl2 or mixtures thereof. The extent of completion of thecoupling reaction can be monitored at each stage, e.g., by the ninhydrinreaction as described by Kaiser et al., Anal. Biochem. 34:595, 1970. Incases where incomplete coupling is found, the coupling reaction isextended and repeated and may have chaotropic salts added. The couplingreactions can be performed automatically with commercially availableinstruments such as ABI model 430A, 431A and 433A peptide synthesizers.

[0122] After the entire assembly of the desired peptide, thepeptide-resin is cleaved with a reagent with proper scavengers. The Fmocpeptides are usually cleaved and deprotected by TFA with scavengers(e.g., H2O, ethanedithiol, phenol and thioanisole). The tboc peptidesare usually cleaved and deprotected with liquid HF for 1-2 hours at −5to 0° C., which cleaves the polypeptide from the resin and removes mostof the side-chain protecting groups. Scavengers such as anisole,dimethylsulfide and p-thiocresol are usually used with the liquid HF toprevent cations formed during the cleavage from alkylating and acylatingthe amino acid residues present in the polypeptide. The formyl group oftryptophan and the dinitrophenyl group of histidine need to be removed,respectively by piperidine and thiophenyl in DMF prior to the HFcleavage. The acetamidomethyl group of cysteine can be removed bymercury(II)acetate and alternatively by iodine,thallium(III)trifluoroacetate or silver tetrafluoroborate whichsimultaneously oxidize cysteine to cystine. Other strong acids used fortBoc peptide cleavage and deprotection include trifluoromethanesulfonicacid (TFMSA) and trimethylsilyltrifluoroacetate (TMSOTf).

[0123] The activity of molecules of the present invention can bemeasured using a variety of assays that measure, for example, cell-cellinteractions, glycolipid and glycoprotein biosynthesis, development, andother biological functions associated with galactosyltransferase familymembers. Of particular interest are changes in the transfer ofgalactosyl molecules in glycoprotein synthesis and in cell-cellinteractions in pancreas, colon, or small intestine tissue cell, linesderived from these tissues. Such assays are well known in the art. For ageneral reference, see Kolbinger, F. et al., J. Biol. Chem. 273:433-440, 1998; Amado, M. et al., J. Biol. Chem. 273:12770-12778, 1998;Hennet, T. et al., J. Biol. Chem. 273:58-65, 1998; and Ram B. P., andMunjal, D. D., CRC Crit. Rev. Biochem. 17:257-311, 1985. Of additionalinterest are differences in cellular expression of znssp2 in diseasedversus non-diseased tissues. Specific assays include but are not limitedto bioassays measuring cell migration, contact inhibition, tissueinteractions, and metastasis. Additional assays would measure neuronalspecificity, fertilization, embryonic cell adhesions, limb budmorphogenesis, mesenchyme development, immune recognition, growthcontrol, tumor metastasis and suppression, and intracellular andextracellular glycoprotein and glycolipid biosynthesis.

[0124] Additional activities likely associated with the polypeptides ofthe present invention include proliferation of cells of the pancreas,colon, spinal cord, bone marrow, small intestine, peripheral leukocytes,bladder, prostate, myometrium, and breast directly or indirectly throughother growth factors; action as a chemotaxic factor; and as a factor forexpanding pancreas and mesenchymal stem cell and precursor populations.

[0125] Another assay of interest measures or detects changes inproliferation, differentiation, and development. Proliferation can bemeasured using cultured primary pancreas cells, ex plant tissues, or invivo by administering molecules of the claimed invention to theappropriate cells, tissues, or animal models. Generally, proliferativeeffects are observed as an increase in cell number and therefore, mayinclude inhibition of apoptosis, as well as mitogenesis. Likewise, adecrease in cell number and cell migration could be analyzed.Established cell lines can be established by one skilled in the art andare available from American Type Culture Collection (Manasas, Va.).Assays measuring cell proliferation are well known in the art. Forexample, assays measuring proliferation include such assays aschemosensitivity to neutral red dye (Cavanaugh et al., InvestigationalNew Drugs 8:347-354, 1990,), incorporation of radiolabelled nucleotides(Cook et al., Analytical Biochem. 179:1-7, 1989,), incorporation of5-bromo-2′-deoxyuridine (BrdU) in the DNA of proliferating cells(Porstmann et al., J. Immunol. Methods 82:169-179, 1985), and use oftetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983; Alley etal., Cancer Res. 48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84,1995; and Scudiero et al., Cancer Res. 48:4827-4833, 1988).

[0126] Proliferation of bone marrow and peripheral blood lymphocytecells can be assayed by harvesting these cells from mice, suspending themononuclear cells in a base medium, and measuring proliferation in thepresence of znssp2 protein. Similarly, clonogenic assays can beperformed.

[0127] To determine if znssp2 is a chemotractant in vivo, znssp2 can begiven by intradermal or intraperitoneal injection. Characterization ofthe accumulated leukocytes at the site of injection can be determinedusing lineage specific cell surface markers and fluorescenceimmunocytometry or by immunohistochemistry (Jose, J. Exp. Med.179:881-87, 1994). Release of specific leukocyte cell populations frombone marrow into peripheral blood can also be measured after znssp2injection.

[0128] Differentiation is a progressive and dynamic process, beginningwith pluripotent stem cells and ending with terminally differentiatedcells. Pluripotent stem cells that can regenerate without commitment toa lineage express a set of differentiation markers that are lost whencommitment to a cell lineage is made. Progenitor cells express a set ofdifferentiation markers that may or may not continue to be expressed asthe cells progress down the cell lineage pathway toward maturation.Differentiation markers that are expressed exclusively by mature cellsare usually functional properties such as cell products, enzymes toproduce cell products and receptors and receptor-like complementarymolecules. The stage of a cell population's differentiation is monitoredby identification of markers present in the cell population. Myocytes,osteoblasts, adipocytes, chrondrocytes, fibroblasts and reticular cellsare believed to originate from a common mesenchymal stem cell (Owen etal., Ciba Fdn. Symp. 136:42-46, 1988). Markers for mesenchymal stemcells have not been well identified (Owen et al., J. of Cell Sci.87:731-738, 1987), so identification is usually made at the progenitorand mature cell stages. The novel polypeptides of the present inventionare useful for studies to isolate mesenchymal stem cells and pancreasprogenitor cells, both in vivo and ex vivo.

[0129] There is evidence to suggest that factors that stimulate specificcell types down a pathway towards terminal differentiation ordedifferentiation affect the entire cell population originating from acommon precursor or stem cell. Thus, znssp2 polypeptides may stimulateinhibition or proliferation of endocrine and exocrine cells of thepancreas, as well as, cells associated with the colon, spinal cord, bonemarrow, heart, small intestine, and peripheral leukocytes. Molecules ofthe present invention may, while stimulating proliferation ordifferentiation of pancreas cells, inhibit proliferation ordifferentiation of other tissues, by virtue of their effect on commonprecursor/stem cells.

[0130] Assays measuring differentiation include, for example, measuringcell-surface markers associated with stage-specific expression of atissue, enzymatic activity, functional activity or morphological changes(Watt, FASEB 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994;Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989).

[0131] The znssp2 polypeptides of the present invention can be used tostudy pancreatic cell proliferation or differentiation. Such methods ofthe present invention generally comprise incubating a cells, α cells, βcells, δ cells and acinar cells in the presence and absence of znssp2polypeptide, monoclonal antibody, agonist or antagonist thereof andobserving changes in cell proliferation or differentiation. An exemplarymodel system to study the formation of pancreatic endocrine cells invitro uses AR42J cells which are derived from acinar cells. Mashima, H.et al., Endocrinology 137:3969-3976, 1996.

[0132] Proteins, including alternatively spliced peptides, andfragments, of the present invention are useful for modulating cell-cellinteractions, neuronal specificity, fertilization, morphogenesis,development, inflammation, tumorigenesis, immune recognition, growthcontrol, tumor suppression, and glycoprotein and glycolipidbiosynthesis. Znssp2 molecules, variants, and fragments can be appliedin isolation, or in conjunction with other molecules (growth factors,cytokines, etc.) in pancreas, colon, spinal cord, bone marrow, heart,small intestine, and peripheral leukocytes. Alternative splicing ofznssp2 may be cell-type specific and confer activity to specifictissues.

[0133] As exemplary cell line of the pancreas to test the activity ofznssp2 is CRL-1682, an human pancreas adenocarcinoma cell line, (ATCC,Manassas, Va.).

[0134] Other assays to measure the effects of znssp2 includeproliferation assays (i.e., of pancreas, bone marrow, spinal cord,colon, or small intestine) by testing tissue and cells from healthyvolunteers with znssp2 protein, or a znssp2-free negative control forthe ability of the tissue and cells to proliferate.

[0135] Proteins of the present invention are useful for delivery oftherapeutic agents such as, but not limited to, radionuclides,chemotherapy agents, and small molecules. The effects of znssp2 can bemeasured in vitro using cultured cells, ex vivo on tissue slices, or invivo by administering molecules of the claimed invention to theappropriate animal model. For instance, znssp2 transfected (orco-transfected) expression host cells may be embedded in an alginateenvironment and injected (implanted) into recipient animals.Alginate-poly-L-lysine microencapsulation, permselective membraneencapsulation and diffusion chambers have been described as a means toentrap transfected mammalian cells or primary mammalian cells. Thesetypes of non-immunogenic “encapsulations” or microenvironments permitthe transfer of nutrients into the microenvironment, and also permit thediffusion of proteins and other macromolecules secreted or released bythe captured cells across the environmental barrier to the recipientanimal. Most importantly, the capsules or microenvironments mask andshield the foreign, embedded cells from the recipient animal's immuneresponse. Such microenvironments can extend the life of the injectedcells from a few hours or days (naked cells) to several weeks (embeddedcells).

[0136] Alginate threads provide a simple and quick means for generatingembedded cells. The materials needed to generate the alginate threadsare readily available and relatively inexpensive. Once made, thealginate threads are relatively strong and durable, both in vitro and,based on data obtained using the threads, in vivo. The alginate threadsare easily manipulable and the methodology is scalable for preparationof numerous threads. In an exemplary procedure, 3% alginate is preparedin sterile H₂O, and sterile filtered. Just prior to preparation ofalginate threads, the alginate solution is again filtered. Anapproximately 50% cell suspension (containing about 5×10⁵ to about 5×10⁷cells/ml) is mixed with the 3% alginate solution. One ml of thealginate/cell suspension is extruded into a 100 mM sterile filteredCaCl₂ solution over a time period of 15 min, forming a “thread”. Theextruded thread is then transferred into a solution of 50 mM CaCl₂, andthen into a solution of 25 mM CaCl₂. The thread is then rinsed withdeionized water before coating the thread by incubating in a 0.01%solution of poly-L-lysine. Finally, the thread is rinsed with LactatedRinger's Solution and drawn from solution into a syringe barrel (withoutneedle attached). A large bore needle is then attached to the syringe,and the thread is intraperitoneally injected into a recipient in aminimal volume of the Lactated Ringer's Solution.

[0137] An alternative in vivo approach for assaying proteins of thepresent invention involves viral delivery systems. Exemplary viruses forthis purpose include adenovirus, herpesvirus, lentivirus, vaccinia virusand adeno-associated virus (AAV). Adenovirus, a double-stranded DNAvirus, is currently the best studied gene transfer vector for deliveryof heterologous nucleic acid (for a review, see T. C. Becker et al.,Meth. Cell Biol. 43:161-89, 1994; and J. T. Douglas and D. T. Curiel,Science & Medicine 4:44-53, 1997). The adenovirus system offers severaladvantages: adenovirus can (i) accommodate relatively large DNA inserts;(ii) be grown to high-titer; (iii) infect a broad range of mammaliancell types; and (iv) be used with a large number of available vectorscontaining different promoters. Also, because adenoviruses are stable inthe bloodstream, they can be administered by intravenous injection.

[0138] By deleting portions of the adenovirus genome, larger inserts (upto 7 kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. In an exemplary system, theessential E1 gene has been deleted from the viral vector, and the viruswill not replicate unless the E1 gene is provided by the host cell (thehuman 293 cell line is exemplary). When intravenously administered tointact animals, adenovirus primarily targets the liver. If theadenoviral delivery system has an E1 gene deletion, the virus cannotreplicate in the host cells. However, the host's tissue (e.g., liver)will express and process (and, if a secretory signal sequence ispresent, secrete) the heterologous protein. Secreted proteins will enterthe circulation in the highly vascularized liver, and effects on theinfected animal can be determined.

[0139] The adenovirus system can also be used for protein production invitro. By culturing adenovirus-infected non-293 cells under conditionswhere the cells are not rapidly dividing, the cells can produce proteinsfor extended periods of time. For instance, BHK cells are grown toconfluence in cell factories, then exposed to the adenoviral vectorencoding the secreted protein of interest. The cells are then grownunder serum-free conditions, which allows infected cells to survive forseveral weeks without significant cell division. Alternatively,adenovirus vector infected 293S cells can be grown in suspension cultureat relatively high cell density to produce significant amounts ofprotein (see Garnier et al., Cytotechnol. 15:145-55, 1994). With eitherprotocol, an expressed, secreted heterologous protein can be repeatedlyisolated from the cell culture supernatant. Within the infected 293Scell production protocol, non-secreted proteins may also be effectivelyobtained.

[0140] In view of the tissue distribution (i.e., pancreas, colon, spinalcord, bone marrow, small intestine, peripheral leukocytes, and variousother tissues) observed for znssp2, agonists (including the naturalligand/substrate/cofactor/etc.) and antagonists have enormous potentialin both in vitro and in vivo applications. Compounds identified asznssp2 agonists are useful for studying galactosylation of cell surfaceantigens as well as cell-cell interactions in vitro and in vivo. Forexample, znssp2 and agonist compounds are useful as components ofdefined cell culture media, and may be used alone or in combination withother cytokines and hormones to replace serum that is commonly used incell culture. Agonists are thus useful in specifically promoting thegrowth and/or development of pancreas, colon, spinal cord, bone marrow,small intestine, and peripheral leukocytes in culture. Alternatively,znssp2 polypeptides and znssp2 agonist polypeptides are useful as aresearch reagent, particularly for the growth and expansion of pancreas,colon or small intestine cells. Znssp2 polypeptides are added to tissueculture media for these cell types.

[0141] Additionally, molecules of the present invention can be used invitro to modify glycoproteins. Aberrant glycosylation can be modified bythe application of the proteins of the present invention. Znssp2molecules can be added in vitro to production or reagent grade proteinsto modify the improper galactosylation of proteins. Additionally,molecules of the present invention can be used in the production ofproperly glycosylated saccharide chains.

[0142] Antagonists are also useful as research reagents forcharacterizing sites of interactions between member ofcomplement/anti-complement pairs as well as site ofgalactosyltransferase catalysis.

[0143] Inhibitors of znssp2 activity (znssp2 antagonists) includeanti-znssp2 antibodies and soluble znssp2 molecules, as well as otherpeptidic and non-peptidic agents (including ribozymes).

[0144] The invention also provides antagonists, which either bind toznssp2 polypeptides or, alternatively, to a anti-complementary moleculeto which znssp2 polypeptides bind, thereby inhibiting or eliminating thefunction of znssp2. Such znssp2 antagonists would include antibodies;polypeptides which bind either to the znssp2 polypeptide or to itsanti-complementary molecule or natural or synthetic analogs of znssp2anti-complementary molecule which retain the ability to bind theanti-complementary molecule but do not result in glycoprotein orglycolipid synthesis or cell-cell interactions. Such analogs could bepeptides or peptide-like compounds. Natural or synthetic small moleculeswhich bind to znssp2 polypeptides and prevent glyprotein or glycolipidsynthesis or cell-cell interactions are also contemplated asantagonists. Also contemplated are soluble znssp2 polypeptides. As such,znssp2 antagonists would be useful as therapeutics for treating certaindisorders where blocking glycosylation or binding of theznssp2-anti-complementary molecule would be beneficial.

[0145] Znssp2 polypeptides may be used within diagnostic systems todetect the presence of znssp2 anti-complementary molecule polypeptides.Antibodies or other agents that specifically bind to znssp2 or itsanti-complementary molecule may also be used to detect the presence ofcirculating znssp2 or anti-complementary molecule polypeptides. Suchdetection methods are well known in the art and include, for example,enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay.Immunohistochemically labeled znssp2 antibodies can be used to detectznssp2 and/or znssp2 anti-complementary molecule in tissue samples.znssp2 levels can also be monitored by such methods as RT-PCR, whereznssp2 mRNA can be detected and quantified. The information derived fromsuch detection methods would provide insight into the significance ofznssp2 polypeptides in various diseases, and as such would serve asdiagnostic tools for diseases for which altered levels of znssp2 aresignificant. Altered levels of znssp2 polypeptides may be indicative ofpathological conditions including, for example, cancer, auto-immunediseases, digestive disordersm and inflammatory disorders.

[0146] A “soluble protein” is a protein that is not bound to a cellmembrane. Soluble proteins are most commonly anti-complementarymolecule-binding polypeptides that lack transmembrane and cytoplasmicdomains. Soluble proteins can comprise additional amino acid residues,such as affinity tags that provide for purification of the polypeptideor provide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface proteinshave naturally occurring, soluble counterparts that are produced byproteolysis or translated from alternatively spliced mRNAs. Proteins aresaid to be substantially free of transmembrane and intracellularpolypeptide segments when they lack sufficient portions of thesesegments to provide membrane anchoring or signal transduction,respectively.

[0147] Soluble forms of znssp2 polypeptides may act as antagonists to oragonists of znssp2 polypeptides, and would be useful to modulate theeffects of znssp2 in pancreas, colon and small intestine. The solubleform of znssp2 does not contain a transmembrane domain (i.e., thepolypeptide of residues 19 to 397 of SEQ ID NO:2) and may act as anagonist or antagonist of znssp2 activity. Since polypeptides of thisnature are not anchored to the membrane, they can act at sites distantfrom the tissues in which they are expressed. Thus, the activity of thesoluble form of znssp2 polypeptides can be more wide spread than itsmembrane-anchored counterpart. Both isoforms would be useful in studyingthe effects of the present invention in vitro an in vivo.

[0148] Znssp2 can also be used to identify inhibitors (antagonists) ofits activity. Test compounds are added to the assays disclosed herein toidentify compounds that inhibit the activity of znssp2. In addition tothose assays disclosed herein, samples can be tested for inhibition ofznssp2 activity within a variety of assays designed to measurecomplementary molecule-anti-complementary molecule binding or thestimulation/inhibition of znssp2-dependent cellular responses. Forexample, znssp2-responsive cell lines can be transfected with a reportergene construct that is responsive to a znssp2-stimulated cellularpathway. Reporter gene constructs of this type are known in the art, andwill generally comprise a znssp2-DNA response-element operably linked toa gene encoding an assayable protein, such as luciferase. DNA responseelements can include, but are not limited to, cyclic AMP responseelements (CRE), hormone response elements (HRE) insulin response element(IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) andserum response elements (SRE) (Shaw et al. Cell 56: 563-72, 1989).Cyclic AMP response elements are reviewed in Roestler et al., J. Biol.Chem. 263 (19):9063-6; 1988 and Habener, Molec. Endocrinol. 4(8):1087-94; 1990. Hormone response elements are reviewed in Beato, Cell56:335-44; 1989. Candidate compounds, solutions, mixtures or extractsare tested for the ability to inhibit the activity of znssp2 on thetarget cells as evidenced by a decrease in znssp2 stimulation ofreporter gene expression. Assays of this type will detect compounds thatdirectly block znssp2 binding to anti-complementary molecules, as wellas compounds that block processes in the cellular pathway subsequent tothis binding. In the alternative, compounds or other samples can betested for direct blocking of znssp2 binding to its anti-complementarymolecule using znssp2 tagged with a detectable label (e.g., ¹²⁵I,biotin, horseradish peroxidase, FITC, and the like). Within assays ofthis type, the ability of a test sample to inhibit the binding oflabeled znssp2 to the anti-complementary molecule is indicative ofinhibitory activity, which can be confirmed through secondary assays.Complementary molecules used within binding assays may be cellularcomplementary molcuels or isolated, immobilized complementary molecules,or receptor-like complementary molecules.

[0149] Assays measuring the inhibition of galactosyltransferase activityin glycoprotein synthesis are listed in Ram, B. P., (ibid).

[0150] Also, znssp2 polypeptides, agonists or antagonists thereof may betherapeutically useful for promoting wound healing, for example, in thepancreas. To verify the presence of this capability in znssp2polypeptides, agonists or antagonists of the present invention, suchznssp2 polypeptides, agonists or antagonists are evaluated with respectto their ability to facilitate wound healing according to proceduresknown in the art. If desired, znssp2 polypeptide performance in thisregard can be compared to growth factors, such as EGF, NGF, TGF-α,TGF-β, insulin, IGF-I, IGF-II, fibroblast growth factor (FGF) and thelike. In addition, znssp2 polypeptides or agonists or antagoniststhereof may be evaluated in combination with one or more growth factorsto identify synergistic effects.

[0151] A znssp2 polypeptide can be expressed as a fusion with animmunoglobulin heavy chain constant region, typically an Fc fragment,which contains two constant region domains and lacks the variableregion. Methods for preparing such fusions are disclosed in U.S. Pat.Nos. 5,155,027 and 5,567,584. Such fusions are typically secreted asmultimeric molecules wherein the Fc portions are disulfide bonded toeach other and two non-Ig polypeptides are arrayed in closed proximityto each other. Fusions of this type can be used to evaluate specificdonor/acceptor molecules, affinity purify ligands, or use as an in vitroassay tool. This fusion can also be used to determine thehomodimerization potential for znssp2. For use in assays, the chimerasare bound to a support via the F_(c) region and used in an ELISA format.

[0152] A znssp2 ligand-binding polypeptide can also be used forpurification of ligand. The polypeptide is immobilized on a solidsupport, such as beads of agarose, cross-linked agarose, glass,cellulosic resins, silica-based resins, polystyrene, cross-linkedpolyacrylamide, or like materials that are stable under the conditionsof use. Methods for linking polypeptides to solid supports are known inthe art, and include amine chemistry, cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, and hydrazide activation. The resulting medium willgenerally be configured in the form of a column or chip, and fluidscontaining ligand are passed through the column or chip one or moretimes to allow ligand to bind to the receptor or receptor-likecomplementary polypeptide. The ligand is then eluted using changes insalt concentration, chaotropic agents (guanidine HCl), or pH to disruptligand-receptor binding.

[0153] An assay system that uses a ligand-binding receptor (or anantibody, one member of a complement/anti-complement pair) or a bindingfragment thereof, and a commercially available biosensor instrument(BIAcore, Pharmacia Biosensor, Piscataway, N.J.) may be advantageouslyemployed. Such receptor, antibody, member of acomplement/anti-complement pair or fragment is immobilized onto thesurface of a receptor chip. Use of this instrument is disclosed byKarlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and Wells,J. Mol. Biol. 234:554-63, 1993. A receptor, antibody, member or fragmentis covalently attached, using amine or sulflhydryl chemistry, to dextranfibers that are attached to gold film within the flow cell. A testsample is passed through the cell. If a ligand, epitope, or oppositemember of the complement/anti-complement pair is present in the sample,it will bind to the immobilized receptor, antibody or member,respectively, causing a change in the refractive index of the medium,which is detected as a change in surface plasmon resonance of the goldfilm. This system allows the determination of on- and off-rates, fromwhich binding affinity can be calculated, and assessment ofstoichiometry of binding.

[0154] Znssp2 polypeptides can also be used within other assay systemsknown in the art. Such systems include Scatchard analysis fordetermination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:660-72, 1949) and calorimetric assays (Cunningham et al., Science253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

[0155] Within the polypeptides of the present invention are polypeptidesthat comprise an epitope-bearing portion of a protein as shown in SEQ IDNOs:2 and 13. An “epitope” is a region of a protein to which an antibodycan bind. See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA81:3998-4002, 1984. Epitopes can be linear or conformational, the latterbeing composed of discontinuous regions of the protein that form anepitope upon folding of the protein. Linear epitopes are generally atleast 6 amino acid residues in length. Relatively short syntheticpeptides that mimic part of a protein sequence are routinely capable ofeliciting an antiserum that reacts with the partially mimicked protein.See, Sutcliffe et al., Science 219:660-666, 1983. Antibodies thatrecognize short, linear epitopes are particularly useful in analytic anddiagnostic applications that employ denatured protein, such as Westernblotting (Tobin, Proc. Natl. Acad. Sci. USA 76:4350-4356, 1979), or inthe analysis of fixed cells or tissue samples. Antibodies to linearepitopes are also useful for detecting fragments of znssp2, such asmight occur in body fluids or cell culture media.

[0156] Antigenic, epitope-bearing polypeptides of the present inventionare useful for raising antibodies, including monoclonal antibodies, thatspecifically bind to a znssp2 protein. The znssp2 polypeptide or afragment thereof serves as an antigen (immunogen) to inoculate an animaland elicit an immune response. One of skill in the art would recognizethat antigenic, epitope-bearing polypeptides contain a sequence of atleast six, or at least nine, or from 15 to about 30 contiguous aminoacid residues of a znssp2 protein (e.g., SEQ ID NO:2). Polypeptidescomprising a larger portion of a znssp2 protein, i.e. from 30 to 100residues up to the entire sequence, are included. Antigens orimmunogenic epitopes can also include attached tags, adjuvants andcarriers, as described herein. Suitable antigens include the znssp2polypeptides 25 encoded by SEQ ID NO:2 from amino acid number 1 to aminoacid number 397, or a contiguous 9 to 397 amino acid fragment thereof.Such regions include secretory sequence, the catalytic domain, or thetransmembrane domain of znssp2 and fragments thereof. Polypeptides inthis regard include those comprising residues 1 to 18 of SEQ ID NO:2;residues 19 to 147 of SEQ ID NO:2; residues 148 to 397 of SEQ ID NO:2;and residues 19 to 397 of SEQ ID NO:2.

[0157] The present invention also provides polypeptide fragments orpeptides comprising an epitope-bearing portion of an znssp2 polypeptidedescribed herein. Such fragments or peptides may comprise an“immunogenic epitope,” which is a part of a protein that elicits anantibody response when the entire protein is used as an immunogen.Immunogenic epitope-bearing peptides can be identified using standardmethods (see, for example, Geysen et al., Proc. Nat'l Acad. Sci. USA81:3998 (1983)).

[0158] In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660(1983)). Accordingly, antigenic epitope-bearing peptides andpolypeptides of the present invention are useful to raise antibodiesthat bind with the polypeptides described herein.

[0159] Antigenic epitope-bearing peptides and polypeptides contain atleast four to ten amino acids, or at least ten to fifteen amino acids,or 15 to 30 amino acids of SEQ ID NOs:2 or 13. Such epitope-bearingpeptides and polypeptides can be produced by fragmenting an znssp2polypeptide, or by chemical peptide synthesis, as described herein.Moreover, epitopes can be selected by phage display of random peptidelibraries (see, for example, Lane and Stephen, Curr. Opin. Immunol.5:268 (1993), and Cortese et al., Curr. Opin. Biotechnol. 7:616 (1996)).Standard methods for identifying epitopes and producing antibodies fromsmall peptides that comprise an epitope are described, for example, byMole, “Epitope Mapping,” in Methods in Molecular Biology, Vol. 10,Manson (ed.), pages 105-116 (The Humana Press, Inc. 1992), Price,“Production and Characterization of Synthetic Peptide-DerivedAntibodies,” in Monoclonal Antibodies: Production, Engineering, andClinical Application, Ritter and Ladyman (eds.), pages 60-84 (CambridgeUniversity Press 1995), and Coligan et al. (eds.), Current Protocols inImmunology, pages 9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley & Sons1997).

[0160] As an illustration, potential antigenic sites in human (SEQ IDNO:2) and mouse (SEQ ID NO:13) znssp2 were identified using theJameson-Wolf method, Jameson and Wolf, CABIOS 4:181, (1988), asimplemented by the PROTEAN program (version 3.14) of LASERGENE (DNASTAR;Madison, Wis.). Default parameters were used in this analysis.

[0161] Suitable antigens of the human sequence include residue 1 toresidue 6 of SEQ ID NO:2; residue 26 to residue 54 of SEQ ID NO:2;residue 82 to residue 94 of SEQ ID NO:2; residue 110 to residue 117 ofSEQ ID NO:2; residue 122 to residue 127 of SEQ ID NO:2; residue 131 toresidue 136 of SEQ ID NO:2; residue 139 to residue 146 of SEQ ID NO:2;residue 154 to residue 177 of SEQ ID NO:2; residue 187 to residue 197 ofSEQ ID NO:2; residue 202 to residue 207 of SEQ ID NO:2; residue 282 toresidue 289 of SEQ ID NO:2; residue 295 to residue 301 of SEQ ID NO:2;residue 358 to residue 365 of SEQ ID NO:2; and residue 387 to residue397 of SEQ ID NO:2; or a portion thereof which contains a 4 to 10 aminoacid segment. Hydrophilic peptides, such as those predicted by one ofskill in the art from a hydrophobicity plot are also immonogenic. Znssp2hydrophilic peptides include peptides comprising amino acid sequencesselected from the group consisting of: residue 24 to residue 53 of SEQID NO:2; residue 72 to residue 81 of SEQ ID NO:2; residue 85 to residue94 of-SEQ ID NO:2; residue 109 to residue 115 of SEQ ID NO:2; residue128 to residue 134 of SEQ ID NO:2; residue 156 to residue 173 of SEQ IDNO:2; residue 200 to residue 209 of SEQ ID NO:2; residue 281 to residue291 of SEQ ID NO:2; residue 297 to residue 306 of SEQ ID NO:2; residue359 to residue 367 of SEQ ID NO:2; and residue 387 to residue 397 of SEQID NO:2; or a portion thereof which contains a 4 to 10 amino acidsegment. Additionally, antigens can be generated to portions of thepolypeptide which are likely to be on the surface of the folded protein.These antigens include: residue 25 to residue 54 of SEQ ID NO:2; residue57 to residue 62 of SEQ ID NO:2; residue 72 to residue 78 of SEQ IDNO:2; residue 84 to residue 93 of SEQ ID NO:2; residue 108 to residue115 of SEQ ID NO:2; residue 155 to residue 167 SEQ ID NO:2; residue 202to residue 207 of SEQ ID NO:2; residue 218 to residue 233 of SEQ IDNO:2; residue 281 to residue 287 of SEQ ID NO:2; residue 358 to residue363 of SEQ ID NO:2; and residue 388 to residue 393 of SEQ ID NO:2; or aportion thereof which contains a 4 to 10 amino acid segment.

[0162] Suitable antigens based on the Jameson-Wolf method for the mousesequence include residue 1 to residue 7 of SEQ ID NO:13; residue 26 toresidue 52 of SEQ ID NO:13; residue 83 to residue 88 of SEQ ID NO:13;residue 125 to residue 132 of SEQ ID NO:13; residue 133 to residue 139of SEQ ID NO:13; residue 146 to residue 152 of SEQ ID NO:13; residue 158to residue 163 of SEQ ID NO:13; residue 183 to residue 189 of SEQ IDNO:13; residue 193 to residue 200 of SEQ ID NO:13; residue 209 toresidue 215 of SEQ ID NO:13; residue 237 to residue 242 of SEQ ID NO:13;residue 273 to residue 281 of SEQ ID NO:13; residue 288 to residue 295of SEQ ID NO:13; residue 351 to residue 360 of SEQ ID NO:13; and residue369 to residue 374 of SEQ ID NO:13; or a portion thereof which containsa 4 to 10 amino acid segment. Hydrophilic peptides, such as thosepredicted by one of skill in the art from a hydrophobicity plot are alsoimmonogenic. znssp2 hydrophilic peptides include peptides comprisingamino acid sequences selected from the group consisting of: residue 1 toresidue 6 of SEQ ID NO:13; residue 24 to residue 53 of SEQ ID NO:13;residue 68 to residue 78 of SEQ ID NO:13; residue 148 to residue 154 ofSEQ ID NO:13; residue 156 ti residue 164 of SEQ ID NO:13; residue 192 toresidue 200 of SEQ ID NO:13; residue 208 to residue 215 of SEQ ID NO:13;residue 273 to residue 280 of SEQ ID NO:13; residue 288 to residue 298of SEQ ID NO:13; and residue 351 to residue 359 of SEQ ID NO: 13; or aportion thereof which contains a 4 to 10 amino acid segment.Additionally, antigens can be generated to portions of the polypeptidewhich are likely to be on the surface of the folded protein. Theseantigens include: residue 25 to residue 37 of SEQ ID NO:13; residue 42to residue 52 of SEQ ID NO:13; residue 69 to residue 76 of SEQ ID NO:13;residue 157 to residue 163 of SEQ ID NO:13; residue 193 to residue 198of SEQ ID NO:13; residue 210 to residue 215 SEQ ID NO:13; residue 271 toresidue 278 of SEQ ID NO:13; and residue 350 to residue 356 of SEQ IDNO:13; or a portion thereof which contains a 4 to 10 amino acid segment.

[0163] Antibodies from an immune response generated by inoculation of ananimal with the antigens listed above can be isolated and purified asdescribed herein.

[0164] Methods for preparing and isolating polyclonal and monoclonalantibodies are well known in the art. See, for example, CurrentProtocols in Immunology, Cooligan, et al. (eds.), National Institutes ofHealth, John Wiley and Sons, Inc., 1995; Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies:Techniques and Applications, CRC Press, Inc., Boca Raton, Fla., 1982.Antibodies generated from this immune response can be isolated andpurified as described herein. Methods for preparing and isolatingpolyclonal and monoclonal antibodies are well known in the art. See, forexample, Current Protocols in Immunology, Cooligan, et al. (eds.),National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrooket al., Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor, N.Y., 1989; and Hurrell, J. G. R., Ed., MonoclonalHybridoma Antibodies: Techniques and Applications, CRC Press, Inc., BocaRaton, Fla., 1982.

[0165] As would be evident to one of ordinary skill in the art,polyclonal antibodies can be generated from inoculating a variety ofwarm-blooded animals such as horses, cows, goats, sheep, dogs, chickens,rabbits, mice, and rats with a znssp2 polypeptide or a fragment thereof.The immunogenicity of a znssp2 polypeptide may be increased through theuse of an adjuvant, such as alum (aluminum hydroxide) or Freund'sadjuvant. Polypeptides useful for immunization also include fusionpolypeptides, such as fusions of znssp2 or a portion thereof with animmunoglobulin polypeptide or with maltose binding protein. Thepolypeptide immunogen may be a full-length molecule or a portionthereof. If the polypeptide portion is “hapten-like”, such portion maybe advantageously joined or linked to a macromolecular carrier (such askeyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanustoxoid) for immunization.

[0166] As used herein, the term “antibodies” includes polyclonalantibodies, affinity-purified polyclonal antibodies, monoclonalantibodies, and antigen-binding fragments, such as F(ab′)₂ and Fabproteolytic fragments. Genetically engineered intact antibodies orfragments, such as chimeric antibodies, Fv fragments, single chainantibodies and the like, as well as synthetic antigen-binding peptidesand polypeptides, are also included. Non-human antibodies may behumanized by grafting non-human CDRs onto human framework and constantregions, or by incorporating the entire non-human variable domains(optionally “cloaking” them with a human-like surface by replacement ofexposed residues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced.

[0167] Alternative techniques for generating or selecting antibodiesuseful herein include in vitro exposure of lymphocytes to znssp2 proteinor peptide, and selection of antibody display libraries in phage orsimilar vectors (for instance, through use of immobilized or labeledznssp2 protein or peptide). Genes encoding polypeptides having potentialznssp2 polypeptide binding domains can be obtained by screening randompeptide libraries displayed on phage (phage display) or on bacteria,such as E. coli. Nucleotide sequences encoding the polypeptides can beobtained in a number of ways, such as through random mutagenesis andrandom polynucleotide synthesis. These random peptide display librariescan be used to screen for peptides which interact with a known targetwhich can be a protein or polypeptide, such as a ligand or receptor, abiological or synthetic macromolecule, or organic or inorganicsubstances. Techniques for creating and screening such random peptidedisplay libraries are known in the art (Ladner et al., U.S. Pat. No.5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S.Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) andrandom peptide display libraries and kits for screening such librariesare available commercially, for instance from Clontech (Palo Alto,Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc.(Beverly, Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway,N.J.). Random peptide display libraries can be screened using the znssp2sequences disclosed herein to identify proteins which bind to znssp2.These “binding proteins” which interact with znssp2 polypeptides can beused for tagging cells; for isolating homolog polypeptides by affinitypurification; they can be directly or indirectly conjugated to drugs,toxins, radionuclides and the like. These binding proteins can also beused in analytical methods such as for screening expression librariesand neutralizing activity. The binding proteins can also be used fordiagnostic assays for determining circulating levels of polypeptides;for detecting or quantitating soluble polypeptides as marker ofunderlying pathology or disease. These binding proteins can also act asznssp2 “antagonists” to block znssp2 binding and signal transduction invitro and in vivo. These anti-znssp2 binding proteins would be usefulfor mediating galactosyltransferase activity extracellularly, therefore,mediating cell-cell interactions, such as, for example, tumor formationand metastasis, proliferation and differentiation, as well asglycoprotein and glycolipid synthesis.

[0168] As used herein, the term “binding proteins” additionally includesantibodies to znssp2 polypeptides, the cognate anti-complementarymolecule of znssp2 polypeptides, proteins useful for purification ofznssp2 polypeptides, and proteins associated with the catalytic(residues 19 to 397 of SEQ ID NO:2).

[0169] Antibodies are determined to be specifically binding if: 1) theyexhibit a threshold level of binding activity, and/or 2) they do notsignificantly cross-react with related polypeptide molecules. First,antibodies herein specifically bind if they bind to a znssp2polypeptide, peptide or epitope with a binding affinity (K_(a)) of 10⁶M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater, more preferably 10⁸ M⁻¹or greater, and most preferably 10⁹ M⁻¹ or greater. The binding affinityof an antibody can be readily determined by one of ordinary skill in theart, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad.Sci. 51: 660-672, 1949).

[0170] Second, antibodies are determined to specifically bind if they donot significantly cross-react with related polypeptides. Antibodies donot significantly cross-react with related polypeptide molecules, forexample, if they detect znssp2 but not known related polypeptides usinga standard Western blot analysis (Ausubel et al., ibid.). Examples ofknown related polypeptides are orthologs, proteins from the same speciesthat are members of a protein family, znssp2 polypeptides, and non-humanznssp2. Moreover, antibodies may be “screened against” known relatedpolypeptides to isolate a population that specifically binds to theinventive polypeptides. For example, antibodies raised to znssp2 areadsorbed to related polypeptides adhered to insoluble matrix; antibodiesspecific to znssp2 will flow through the matrix under the proper bufferconditions. Such screening allows isolation of polyclonal and monoclonalantibodies non-crossreactive to closely related polypeptides(Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988; Current Protocols in Immunology,Cooligan, et al. (eds.), National Institutes of Health, John Wiley andSons, Inc., 1995). Screening and isolation of specific antibodies iswell known in the art. See, Fundamental Immunology, Paul (eds.), RavenPress, 1993; Getzoffet al., Adv. in Immunol. 43: 1-98, 1988; MonoclonalAntibodies: Principles and Practice, Goding, J. W. (eds.), AcademicPress Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984.

[0171] A variety of assays known to those skilled in the art can beutilized to detect antibodies which specifically bind to znssp2 proteinsor peptides. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutant znssp2protein or polypeptide.

[0172] Antibodies to znssp2 may be used for tagging cells that expressznssp2; for isolating znssp2 by affinity purification; in analyticalmethods employing FACS; for screening expression libraries; forgenerating anti-idiotypic antibodies; and as neutralizing antibodies oras antagonists to block znssp2 in vitro and in vivo. Suitable directtags or labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent markers, chemiluminescent markers, magneticparticles and the like; indirect tags or labels may feature use ofbiotin-avidin or other complement/anti-complement pairs asintermediates. Antibodies herein may also be directly or indirectlyconjugated to drugs, toxins, radionuclides and the like, and theseconjugates used for in vivo diagnostic or therapeutic applications.Moreover, antibodies to znssp2 or fragments thereof may be used in vitroto detect denatured znssp2 or fragments thereof in assays, for example,Western Blots or other assays known in the art.

[0173] The soluble znssp2 is useful in studying the distribution of itsanti-complentary molecule in tissues or specific cell lineages, and toprovide insight into complementary molecule-anti-complentary moleculebiology. Using labeled znssp2, cells expressing the anti-complentarymolecule are identified by fluorescence immunocytometry orimmunocytochemistry. Application may also be made of 7 the specificityof UDP-glycosyltransferases for their substrates.

[0174] Antibodies can be made to soluble, znssp2 polypeptides which areHis or FLAG™ tagged. Alternatively, such polypeptides form a fusionprotein with Human Ig. In particular, antiserum containing polypeptideantibodies to His-tagged, or FLAG™-tagged soluble znssp2 can be used inanalysis of tissue distribution of znssp2 by immunohistochemistry onhuman or primate tissue. These soluble znssp2 polypeptides can also beused to immunize mice in order to produce monoclonal antibodies to asoluble human znssp2 polypeptide. Monoclonal antibodies to a solublehuman znssp2 polypeptide can also be used to mimic anti-complentarymolecule coupling, resulting in activation or inactivation of thecomplementary molecule-anti-complentary molecule pair. For instance, ithas been demonstrated that cross-linking anti-soluble CD40 monoclonalantibodies provides a stimulatory signal to B cells that have beensub-optimally activated with anti-IgM or LPS, and results inproliferation and immunoglobulin production. These same monoclonalantibodies act as antagonists when used in solution by blockingactivation of the receptor. Monoclonal antibodies to znssp2 can be usedto determine the distribution, regulation and biological interaction ofthe znssp2 and its anti-complentary molecule pair on specific celllineages identified by tissue distribution studies.

[0175] Antibodies or polypeptides herein can also be directly orindirectly conjugated to drugs, toxins, radionuclides and the like, andthese conjugates used for in vivo diagnostic or therapeuticapplications. For instance, polypeptides or antibodies of the presentinvention can be used to identify or treat tissues or organs thatexpress a corresponding anti-complementary molecule (receptor, enzyme,receptor-like complementary molecule or antigen, respectively, forinstance). More specifically, znssp2 polypeptides or anti-znssp2antibodies, or bioactive fragments or portions thereof, can be coupledto detectable or cytotoxic molecules and delivered to a mammal havingcells, tissues or organs that express the anti-complementary molecule.

[0176] Suitable detectable molecules may be directly or indirectlyattached to the polypeptide or antibody, and include radionuclides,enzymes, substrates, cofactors, inhibitors, fluorescent markers,chemiluminescent markers, magnetic particles and the like. Suitablecytotoxic molecules may be directly or indirectly attached to thepolypeptide or antibody, and include bacterial or plant toxins (forinstance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and thelike), as well as therapeutic radionuclides, such as iodine-131,rhenium-188 or yttrium-90 (either directly attached to the polypeptideor antibody, or indirectly attached through means of a chelating moiety,for instance). Polypeptides or antibodies may also be conjugated tocytotoxic drugs, such as adriamycin. For indirect attachment of adetectable or cytotoxic molecule, the detectable or cytotoxic moleculecan be conjugated with a member of a complementary/anticomplementarypair, where the other member is bound to the polypeptide or antibodyportion. For these purposes, biotin/streptavidin is an exemplarycomplementary/anticomplementary pair.

[0177] In another embodiment, polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer or diseased cellsor tissues). Alternatively, if the polypeptide has multiple functionaldomains (i.e., an activation domain or a ligand binding domain, plus atargeting domain), a fusion protein including only the targeting domainmay be suitable for directing a detectable molecule, a cytotoxicmolecule or a complementary molecule to a cell or tissue type ofinterest. In instances where the domain only fusion protein includes acomplementary molecule, the anti-complementary molecule can beconjugated to a detectable or cytotoxic molecule. Suchdomain-complementary molecule fusion proteins thus represent a generictargeting vehicle for cell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

[0178] In another embodiment, znssp2-cytokine fusion proteins orantibody-cytokine fusion proteins can be used for enhancing in vivokilling of target tissues (for example, pancreas, colon, spinal cord,bone marrow, small intestine and peripheral leukocyte cancers), if theznssp2 polypeptide or anti-znssp2 antibody targets, for example, thehyperproliferative pancreas, colon, spinal cord, bone marrow, smallintestine and peripheral leukocyte cells (See, generally, Hornick etal., Blood 89:4437-47, 1997). They described fusion proteins enabletargeting of a cytokine to a desired site of action, thereby providingan elevated local concentration of cytokine. Suitable znssp2polypeptides or anti-znssp2 antibodies target an undesirable cell ortissue (i.e., a tumor or a leukemia), and the fused cytokine mediatedimproved target cell lysis by effector cells. Suitable cytokines forthis purpose include interleukin 2 and granulocyte-macrophagecolony-stimulating factor (GM-CSF), for instance.

[0179] In yet another embodiment, if the znssp2 polypeptide oranti-znssp2 antibody targets vascular cells or tissues, such polypeptideor antibody may be conjugated with a radionuclide, and particularly witha beta-emitting radionuclide, to reduce restenosis. Such therapeuticapproach poses less danger to clinicians who administer the radioactivetherapy. The bioactive polypeptide or antibody conjugates describedherein can be delivered intravenously, intraarterially or intraductally,or may be introduced locally at the intended site of action.

[0180] The bioactive polypeptide or antibody conjugates described hereincan be delivered intravenously, intraarterially or intraductally, or maybe introduced locally at the intended site of action.

[0181] znssp2 polynucleotides and/or polypeptides may be useful forregulating the maturation of UDP-glycosyltransferase substrate-bearingcells, such as fibroblasts, lymphocytes and hematopoietic cells. znssp2polypeptides will also find use in mediating metabolic or physiologicalprocesses in vivo. The effects of a compound on proliferation anddifferentiation can be measured in vitro using cultured cells. Bioassaysand ELISAs are available to measure cellular response to znssp2, inparticular are those which measure changes in cytokine production as ameasure of cellular response (see for example, Current Protocols inImmunology ed. John E. Coligan et al., NIH, 1996). Assays to measureother cellular responses, including glycoprotein and glycolipidbiosynthesis and metabolism, and cell-cell interactions are known in theart.

[0182] The activity of znssp2 or a peptide to which znssp2 binds, can bemeasured by a silicon-based biosensor microphysiometer which measuresthe extracellular acidification rate or proton excretion associated withsuch protein interactions and subsequent physiologic cellular responses.An exemplary device is the Cytosensor™ Microphysiometer manufactured byMolecular Devices, Sunnyvale, Calif. A variety of cellular responses,such as cell proliferation, ion transport, energy production,inflammatory response, regulatory and enzyme or enzyme activation, andthe like, can be measured by this method. See, for example, McConnell,H. M. et al., Science 257:1906-1912, 1992; Pitchford, S. et al., Meth.Enzymol. 228:84-108, 1997; Arimilli, S. et al., J. Immunol. Meth.212:49-59, 1998; Van Liefde, I. et al., Eur. J. Pharmacol. 346:87-95,1998. The microphysiometer can be used for assaying adherent ornon-adherent eukaryotic or prokaryotic cells. By measuring extracellularacidification changes in cell media over time, the microphysiometerdirectly measures cellular responses to various stimuli, includingznssp2 proteins, their agonists, and antagonists. The microphysiometercan be used to measure responses of a znssp2-responsive eukaryotic cell,compared to a control eukaryotic cell that does not respond to znssp2polypeptide. znssp2-responsive eukaryotic cells comprise cells intowhich a polynucleotide for znssp2 has been transfected creating a cellthat is responsive to znssp2; or cells containing endogenous znssp2polynucleotides. Differences, measured by a change in the response ofcells exposed to znssp2 anti-complentary molecule, relative to a controlnot exposed to znssp2 anti-complentary molecule, directly measure theznssp2-modulated cellular responses. Moreover, such znssp2-modulatedresponses can be assayed under a variety of stimuli. The presentinvention provides a method of identifying agonists and antagonists ofznssp2 protein, comprising providing cells responsive to a znssp2polypeptide, culturing a first portion of the cells in the absence of atest compound, culturing a second portion of the cells in the presenceof a test compound, and detecting a measurable change in a cellularresponse of the second portion of the cells as compared to the firstportion of the cells. Moreover, culturing a third portion of the cellsin the presence of znssp2 substrate and the absence of a test compoundprovides a positive control for the znssp2-responsive cells, and acontrol to compare the agonist activity of a test compound with that ofthe znssp2 substrate. Antagonists of znssp2 can be identified byexposing the cells to znssp2 substrate in the presence and absence ofthe test compound, whereby a reduction in znssp2-modulated activity isindicative of antagonist activity in the test compound.

[0183] Moreover, znssp2 can be used to identify cells, tissues, or celllines which respond to a znssp2-modulated pathway. The microphysiometer,described above, can be used to rapidly identify cells responsive toznssp2 of the present invention. Cells can be cultured in the presenceor absence of znssp2 polypeptide. Those cells which elicit a measurablechange in extracellular acidification in the presence of znssp2 areresponsive to znssp2. Such cell lines, can be used to identify znssp2anti-complentary molecule, antagonists and agonists of znssp2polypeptide as described above.

[0184] Molecules of the present invention can be used to identify andisolate receptors, ligands, or members of complement/anti-complementpairs involved in cell-cell interactions, and glycoprotein andglycolipid synthesis. For example, proteins and peptides of the presentinvention can be immobilized on a column and membrane preparations runover the column (Immobilized Affinity Ligand Techniques, Hermanson etal., eds., Academic Press, San Diego, Calif., 1992, pp.195-202).Proteins and peptides can also be radiolabeled (Methods in Enzymol.,vol. 182, “Guide to Protein Purification”, M. Deutscher, ed., Acad.Press, San Diego, 1990, 721-37) or photoaffinity labeled (Brunner etal., Ann. Rev. Biochem. 62:483-514, 1993 and Fedan et al., Biochem.Pharmacol. 33:1167-80, 1984) and specific cell-surface proteins can beidentified.

[0185] As a reagent, the polynucleotide encoding the amino acid residuesfrom residue 333 to 338 of SEQ ID NO: 2, and the degeneratepolynucleotide of SEQ ID NO:3, can be used to identify new familymembers. This would be useful in finding new galactosyltransferase andputative neurogenic secreted signaling peptides from the same or othertissues.

[0186] The polypeptides, nucleic acid and/or antibodies of the presentinvention can be used in treatment of disorders associated with cellmigration, contact inhibition, tissue interactions, neuronalspecificity, fertilization, embryonic cell adhesions, limb budmorphogenesis, mesenchyme development, immune recognition, inflammation,tumorigenesis, growth control, tumor metastasis, and intracellular andextracellular glycoprotein and glycolipid biosynthesis. The molecules ofthe present invention can be used to modulate glycoprotein synthesisand/or cell-cell interactions or to treat or prevent development ofpathological conditions in such diverse tissue as pancreas, colon,spinal cord, bone marrow, heart, lung, spleen, prostate, smallintestine, peripheral blood leukocytes, stomach, thyroid, trachea,placenta, skeletal muscle, kidney, lymph node, bladder, prostate,myometrium, spleen, and breast. In particular, certain pancreaticenzymatic deficiencies and malignancies, and pancreatic-cell mediateddeficiencies may be amenable to such diagnosis, treatment or prevention.

[0187] Polynucleotides encoding znssp2 polypeptides are useful withingene therapy applications where it is desired to increase or inhibitznssp2 activity. If a mammal has a mutated or absent znssp2 gene, theznssp2 gene can be introduced into the cells of the mammal. In oneembodiment, a gene encoding a znssp2 polypeptide is introduced in vivoin a viral vector. Such vectors include an attenuated or defective DNAvirus, such as, but not limited to, herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associatedvirus (AAV), and the like. Defective viruses, which entirely or almostentirely lack viral genes, are preferred. A defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Examples ofparticular vectors include, but are not limited to, a defective herpessimplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci.2:320-30, 1991); an attenuated adenovirus vector, such as the vectordescribed by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30,1992; and a defective adeno-associated virus vector (Samulski et al., J.Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).

[0188] In another embodiment, a znssp2 gene can be introduced in aretroviral vector, e.g., as described in Anderson et al., U.S. Pat. No.5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No.4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J.Virol. 62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263;International Patent Publication No. WO 95/07358, published Mar. 16,1995 by Dougherty et al.; and Kuo et al., Blood 82:845, 1993.

[0189] Alternatively, the vector can be introduced by lipofection invivo using liposomes. Synthetic cationic lipids can be used to prepareliposomes for in vivo transfection of a gene encoding a marker (Felgneret al., Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc.Natl. Acad. Sci. USA 85:8027-31, 1988). The use of lipofection tointroduce exogenous genes into specific organs in vivo has certainpractical advantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. More particularly, directingtransfection to particular cells represents one area of benefit. Forinstance, directing transfection to particular cell types would beparticularly advantageous in a tissue with cellular heterogeneity, suchas the pancreas, liver, kidney, and brain. Lipids may be chemicallycoupled to other molecules for the purpose of targeting. Targetedpeptides (e.g., hormones or neurotransmitters), proteins such asantibodies, or non-peptide molecules can be coupled to liposomeschemically. Similarly, the znssp2 polynucleotide itself can be used totarget specific tissues.

[0190] It is possible to remove the target cells from the body; tointroduce the vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

[0191] Various techniques, including antisense and ribozymemethodologies, can be used to inhibit znssp2 gene transcription andtranslation, such as to inhibit cell proliferation in vivo.Polynucleotides that are complementary to a segment of a znssp2-encodingpolynucleotide (e.g., a polynucleotide as set froth in SEQ ID NOs:1 or12) are designed to bind to znssp2-encoding mRNA and to inhibittranslation of such mRNA. Such antisense polynucleotides are used toinhibit expression of znssp2 polypeptide-encoding genes in cell cultureor in a subject.

[0192] The present invention also provides reagents which will find usein diagnostic applications. For example, the znssp2 gene, a probecomprising znssp2 DNA or RNA or a subsequence thereof can be used todetermine if the znssp2 gene is present on chromosome 19q13.2 or if amutation has occurred. Detectable chromosomal aberrations at the znssp2gene locus include, but are not limited to, aneuploidy, gene copy numberchanges, insertions, deletions, restriction site changes andrearrangements. These aberrations can occur within the coding sequence,within introns, or within flanking sequences, including upstreampromoter and regulatory regions, and may be manifested as physicalalterations within a coding sequence or changes in gene expressionlevel. Such aberrations can be detected using polynucleotides of thepresent invention by employing molecular genetic techniques, such asrestriction fragment length polymorphism (RFLP) analysis, short tandemrepeat (STR) analysis employing PCR techniques, and other geneticlinkage analysis techniques known in the art (Sambrook et al., ibid;Ausubel et. al., ibid; Marian, Chest 108:255-65, 1995).

[0193] In general, these diagnostic methods comprise the steps of (a)obtaining a genetic sample from a patient; (b) incubating the geneticsample with a polynucleotide probe or primer as disclosed above, underconditions wherein the polynucleotide will hybridize to complementarypolynucleotide sequence, to produce a first reaction product; and (iii)comparing the first reaction product to a control reaction product. Adifference between the first reaction product and the control reactionproduct is indicative of a genetic abnormality in the patient. Geneticsamples for use within the present invention include genomic DNA, cDNA,and RNA. The polynucleotide probe or primer can be RNA or DNA, and willcomprise a portion of SEQ ID NOs:1 or 3, the complement of SEQ ID NOs:1or 3, or an RNA equivalent thereof. Suitable assay methods in thisregard include molecular genetic techniques known to those in the art,such as restriction fragment length polymorphism (RFLP) analysis, shorttandem repeat (STR) analysis employing PCR techniques, ligation chainreaction (Barany, PCR Methods and Applications 1:5-16, 1991),ribonuclease protection assays, and other genetic linkage analysistechniques known in the art (Sambrook et al., ibi; Ausubel et. al.,ibid.; Marian, Chest 108:255-65, 1995). Ribonuclease protection assays(see, e.g., Ausubel et al., ibid., ch. 4) comprise the hybridization ofan RNA probe to a patient RNA sample, after which the reaction product(RNA-RNA hybrid) is exposed to RNase. Hybridized regions of the RNA areprotected from digestion. Within PCR assays, a patient's genetic sampleis incubated with a pair of polynucleotide primers, and the regionbetween the primers is amplified and recovered. Changes in size oramount of recovered product are indicative of mutations in the patient.Another PCR-based technique that can be employed is single strandconformational polymorphism (SSCP) analysis (Hayashi, PCR Methods andApplications 1:34-8, 1991).

[0194] In addition, such polynucleotide probes could be used tohybridize to counterpart sequences on individual chromosomes.Chromosomal identification and/or mapping of the znssp2 gene couldprovide useful information about gene function and disease association.Many mapping techniques are available to one skilled in the art, forexample, mapping somatic cell hybrids, and fluorescence in situhybridization (FISH). One method is radiation hybrid mapping. Radiationhybrid mapping is a somatic cell genetic technique developed forconstructing high-resolution, contiguous maps of mammalian chromosomes(Cox et al., Science 250:245-50, 1990). Partial or full knowledge of agene's sequence allows one to design PCR primers suitable for use withchromosomal radiation hybrid mapping panels. Radiation hybrid mappingpanels are commercially available which cover the entire human genome,such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (ResearchGenetics, Inc., Huntsville, Ala.). These panels enable rapid, PCR-basedchromosomal localizations and ordering of genes, sequence-tagged sites(STSs), and other nonpolymorphic and polymorphic markers within a regionof interest. This includes establishing directly proportional physicaldistances between newly discovered genes of interest and previouslymapped markers. The precise knowledge of a gene's position can be usefulfor a number of purposes, including: 1) determining if a sequence ispart of an existing contig and obtaining additional surrounding geneticsequences in various forms, such as YACs, BACs or cDNA clones; 2)providing a possible candidate gene for an inheritable disease whichshows linkage to the same chromosomal region; and 3) cross-referencingmodel organisms, such as mouse, which may aid in determining whatfunction a particular gene might have.

[0195] Sequence tagged sites (STSs) can also be used independently forchromosomal localization. An STS is a DNA sequence that is unique in thehuman genome and can be used as a reference point for a particularchromosome or region of a chromosome. An STS is defined by a pair ofoligonucleotide primers that are used in a polymerase chain reaction tospecifically detect this site in the presence of all other genomicsequences. Since STSs are based solely on DNA sequence they can becompletely described within an electronic database, for example,Database of Sequence Tagged Sites (dbSTS), GenBank, (National Center forBiological Information, National Institutes of Health, Bethesda, Md.http://www.ncbi.nlm.nih.gov), and can be searched with a gene sequenceof interest for the mapping data contained within these short genomiclandmark STS sequences.

[0196] Transgenic mice, engineered to express the znssp2 gene, and micethat exhibit a complete absence of znssp2 gene function, referred to as“knockout mice” (Snouwaert et al., Science 257:1083, 1992), may also begenerated (Lowell et al., Nature 366:740-42, 1993). These mice may beemployed to study the znssp2 gene and the protein encoded thereby in anin vivo system.

[0197] Znssp2 polypeptides, variants, and fragments thereof, may beuseful as replacement therapy for disorders associated with glycoproteinsynthesis, functions of the digestive system, and cell-cellinteractions.

[0198] A less widely appreciated determinant of tissue morphogenesis isthe process of cell rearrangement: Both cell motility and cell-celladhesion are likely to play central roles in morphogenetic cellrearrangements. Cells need to be able to rapidly break and probablysimultaneously remake contacts with neighboring cells. See Gumbiner, B.M., Cell 69:385-387, 1992. As a secreted protein in tissues of thepancreas, colon, small intestine, etc., znssp2 can play a role inintercellular rearrangement in these and other tissues.

[0199] The znssp2 polypeptide is expressed in tissues of the pancreas,colon, spinal cord, bone marrow, small intestine, and peripheralleukocytes. Thus, the polypeptides of the present invention are usefulin studying cell adhesion and the role thereof in metastasis and may beuseful in preventing metastasis, in particular metastasis in tumors ofthe pancreas, colon, spinal cord, bone marrow, small intestine, andperipheral leukocytes. Similarly, polynucleotides and polypeptides ofznssp2 may be used to replace their defective counterparts in tumor ordiseased tissues. Thus, znssp2 polypeptide pharmaceutical compositionsof the present invention may be useful in prevention or treatment ofdisorders associated with pathological regulation or the expansion ofthese tissues. The polynucleotides of the present invention may also beused in conjunction with a regulatable promoter, thus allowing thedosage of delivered protein to be regulated.

[0200] Moreover, the activity and effect of znssp2 on tumor progressionand metastasis can be measured in vivo. Several syngeneic mouse modelshave been developed to study the influence of polypeptides, compounds orother treatments on tumor progression. In these models, tumor cellspassaged in culture are implanted into mice of the same strain as thetumor donor. The cells will develop into tumors having similarcharacteristics in the recipient mice, and metastasis will also occur insome of the models. Tumor models include the Lewis lung carcinoma (ATCCNo. CRL-1642) and B 16 melanoma (ATCC No. CRL-6323), amongst others.These are both commonly used tumor lines, syngeneic to the C57BL6 mouse,that are readily cultured and manipulated in vitro. Tumors resultingfrom implantation of either of these cell lines are capable ofmetastasis to the lung in C57BL6 mice. The Lewis lung carcinoma modelhas recently been used in mice to identify an inhibitor of angiogenesis(O'Reilly M S, et al. Cell 79: 315-328,1994). C57BL6/J mice are treatedwith an experimental agent either through daily injection of recombinantprotein, agonist or antagonist or a one time injection of recombinantadenovirus. Three days following this treatment, 10⁵ to 10⁶ cells areimplanted under the dorsal skin. Alternatively, the cells themselves maybe infected with recombinant adenovirus, such as one expressing znssp2,before implantation so that the protein is synthesized at the tumor siteor intracellularly, rather than systemically. The mice normally developvisible tumors within 5 days. The tumors are allowed to grow for aperiod of up to 3 weeks, during which time they may reach a size of1500-1800 mm³ in the control treated group. Tumor size and body weightare carefully monitored throughout the experiment. At the time ofsacrifice, the tumor is removed and weighed along with the lungs and theliver. The lung weight has been shown to correlate well with metastatictumor burden. As an additional measure, lung surface metastases arecounted. The resected tumor, lungs and liver are prepared forhistopathological examination, immunohistochemistry, and in situhybridization, using methods known in the art and described herein. Theinfluence of the expressed polypeptide in question, e.g., znssp2, on theability of the tumor to recruit vasculature and undergo metastasis canthus be assessed. In addition, aside from using adenovirus, theimplanted cells can be transiently transfected with znssp2. Moreover,purified znssp2 or znssp2-conditioned media can be directly injected into this mouse model, and hence be used in this system. Use of stableznssp2 transfectants as well as use of induceable promoters to activateznssp2 expression in vivo are known in the art and can be used in thissystem to assess znssp2 induction of metastasis. For general referencesee, O'Reilly M S, et al. Cell 79:315-328, 1994; and Rusciano D, et al.Murine Models of Liver Metastasis. Invasion Metastasis 14:349-361, 1995.

[0201] Tn-syndrome, also called Permanent Mixed-FieldPolyagglutinability, is a very rare acquired disorder affecting allhematopoietic lineages. This syndrome is characterized by the expressionof the Tn and sialosyl-Tn antigens on the cell surface. The Tn antigenhas been identified as an unsubstituted α-linked N-acetyl-galactosaminelinked O-glycosidically to threonine or serine residues of membraneproteins. In healthy blood, this sugar is substituted by galactose andsialic acid to form a tetrasaccharide. This Tn antigen may be a resultof a deficiency in β1

3,galactosyltransferase. Expression of the Tn antigen along with thesialosyl-Tn antigen and a TF antigen (characterized by a deficiency inα,2

3,sialyl-transferase) have been recognized as a cancer-associatedphenomenon for many years. See Berger, E. G. et al., Transfus. Clin.Biol. 2:103-108, 1994.

[0202] Thus, the study of this syndrome has been useful in elucidatingthe biology of carbohydrate glycosylation disorders and the appearanceof cryptantigens on the cell surface, and cancer. Highly specific andcomplex tumor glycan antigens are likely of great interest in studyingtissue specific tumors and znssp2 can be useful for studying tumors ofthe pancreas, colon, spinal cord, bone marrow, small intestine, andperipheral leukocytes.

[0203] Itzkowitz, et al., looked at the expression of thesecryptantigens in tissues from normal, chronic pancreatitic, andpancreatic cancer patients. The sialosyl-Tn antigen is expressed in 97%of malignant, but 0% of normal tissues. The authors suggest that normalpancreas tissue is preferentially galactosylated resulting in lesssilaosyl-Tn antigen. In malignant tissue, conditions favor thesialylation of Tn antigens thereby accounting for enhanced expression ofsialosyl Tn over T anitgens.

[0204] In view of the high expression of znssp2 in the pancreas, andcolon in normal tissue, a defect in the znssp2 gene may result indefective galactosylation of cell surface carbohydrates of pancreaticcells, leading to over sialylation of the Tn antigen, or overgalactosylation of cellular antigens, in general. Thus, znssp2polypeptides would be useful as a pancreas- or colon-specific β,1

3, galactosyltransferase replacement therapy for pre-cancerous andcancer tissues. To verify the presence of such activity in znssp2containing normal cell lines and tumor cell lines, such cell lines areevaluated with respect to the presence of the Tn antigen according toprocedures known in the art. See, for example, Berger et al., ibid.,Itzkowitz et al., ibid. and the like.

[0205] Additionally, the lack of conditions favoring propergalactosylation may result in an increase in sialosyl Tn antigens intissues expressing znssp2, which may cause an auto-immune reactionresulting in an immune attack on the pancreas, colon, spinal cord, bonemarrow, small intestine, and peripheral leukocytes. In these cases,znssp2 molecules may be used to encourage proper galactosylation andlimit the antigenic recognition in tissues over expressing the sialosylTn antigen.

[0206] Similarly, a defective znssp2 gene may result in improperglycoslation of the surface carbohydrates of the tissues of pancreas,colon, spinal cord, bone marrow, small intestine, and peripheralleukocytes, thus affecting cell-cell interactions and possibly cellcycle regulation. Such cases could be treated by administeringpolypeptides of znssp2 to mammals with such a defective gene.

[0207] Exocrine cells of the pancreas are important for the productionof necessary enzymes involved in digestion. Persons defective in theznssp2 gene may be unable to properly digest food and and nutrients.Polynucleotides of znssp2 may be useful in treating a defectivepancreatic specific β,1

3,galactosyl-transferase gene by gene therapy. Likewise, polypeptides ofthe present invention could be administered to a mammal as replacementtherapy for a defective digestive enzyme.

[0208] Znssp2 gene may be useful to as a probe to identify humans whohave a defective pancreatic or colonic specific β,1

3, galactosyltransferase gene. The strong expression of znssp2 inpancreas, and colon suggests that znssp2 polynucleotides or polypeptidesare down-regulated in tumor, malignant, or immune-responding tissues.Thus, polynucleotides and polypeptides of znssp2, and mutations to them,can be used a indicators of pancreatic and colonic cancer, and disease,in diagnosis.

[0209] As a protein showing strong expression in the pancreas and colon,additional applications are to modulate gastric secretion in thetreatment of acute pancreatitis and gastrointestinal disorders.

[0210] The polypeptides, nucleic acid, and/or antibodies of the presentinvention may be used in treatment of disorders associated withpancreas, diabetes, hypoglycemia; digestive systems including pancreas,colon, and small intestine; neuronal tissues, bone marrow, andperipheral leukocytes; and in disorders associated with glycoproteinsynthesis. The molecules of the present invention may used to modulateor to treat or prevent development of pathological conditions in suchdiverse tissue as pancreas, colon, spinal cord, heart and bone marrow.In particular, certain syndromes or diseases may be amenable to suchdiagnosis, treatment or prevention.

[0211] The znssp2 polypeptide is expressed in the pancreas. Thus, znssp2polypeptide pharmaceutical compositions of the present invention may beuseful in prevention or treatment of pancreatic disorders associatedwith pathological regulation of the expansion of neuroendocrine andexocrine cells in the pancreas, such as IDDM, pancreatic cancer,pathological regulation of blood glucose levels, insulin expression,insulin resistance or digestive function.

[0212] The znssp2 polypeptide of the present invention may act in theneuroendocrine/exocrine cell fate decision pathway and may therefore becapable of regulating the expansion of neuroendocrine and exocrine cellsin the pancreas. One such regulatory use is that of islet cellregeneration. Also, it has been hypothesized that the autoimmunity thattriggers IDDM starts in utero, and znssp2 polypeptide is a developmentalgene involved in cell partitioning. Assays and animal models are knownin the art for monitoring the exocrine/neuroendocrine cell lineagedecision, for observing pancreatic cell balance and for evaluatingznssp2 polypeptide, fragment, fusion protein, antibody, agonist orantagonist in the prevention or treatment of the conditions set forthabove.

[0213] For pharmaceutical use, the proteins of the present invention areformulated for parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods. Intravenous administrationwill be by bolus injection or infusion over a typical period of one toseveral hours. In general, pharmaceutical formulations will include aznssp2 protein in combination with a pharmaceutically acceptablevehicle, such as saline, buffered saline, 5% dextrose in water or thelike. Formulations may further include one or more excipients,preservatives, solubilizers, buffering agents, albumin to preventprotein loss on vial surfaces, etc. Methods of formulation are wellknown in the art and are disclosed, for example, in Remington: TheScience and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. Therapeutic doses will generally be in therange of 0.1 to 100 μg/kg of patient weight per day, preferably 0.5-20mg/kg per day, with the exact dose determined by the clinician accordingto accepted standards, taking into account the nature and severity ofthe condition to be treated, patient traits, etc. Determination of doseis within the level of ordinary skill in the art. The proteins may beadministered for acute treatment, over one week or less, often over aperiod of one to three days or may be used in chronic treatment, overseveral months or years. In general, a therapeutically effective amountof znssp2 is an amount sufficient to produce a clinically significantchange in disorders such as, diabetes, autoimmunity, cancer, as well asdisorders of the digestive system including, Crohn's disease, ulcerativecolitis, pancreatitis, digestive enzymatic disfunction, and cancersuppression and ablation. Similarly, a therapeutically effective amountof znssp2 is an amount sufficient to produce a clinically significantchange in disorders accociated with heart, placenta, lung, skeletonmuscle, kidney, spleen, prostate, small intestine, colon, peripheralleukocyte, stomach, thyroid, spinal cord, lymph node, trachea, bonemarrow, bladder, breast, prostate, and myometrium.

[0214] The invention is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1

[0215] Extension of EST Sequence

[0216] The novel znssp2 polypeptide-encoding polynucleotides of thepresent invention were initially identified by querying an EST database.A cDNA clone, corresponding to an EST was obtained and the deduced aminoacid sequence was determined to be incomplete at the 5′ terminal. Nested5′ RACE polymerase chain reactions were performed. The first RACE usedprimers ZC9719 (SEQ ID NO:8) and ZC17035 (SEQ ID NO:9) and thermolcyclerconditions as follows: one cycle at 94° C. for 2 minutes; followed bytwenty-five cycles at 94° C. for 20 seconds, 65° C. for 30 seconds, 72°C. for 45 seconds, followed by one cycle at 72° C. for 2 minutes. Bonemarrow marathon cDNA was used as a template. The second, nested, RACEreaction used primers ZC9739 (SEQ ID NO:10) and ZC17036 (SEQ ID NO:11)and thermolcycler conditions as follows: one cycle at 94° C. for 2minutes; followed by five cycles at 94° C. for 20 seconds, 69° C. for45; followed by twenty-eight cycles 94° C. for 20 seconds, 64° C. for 30seconds, 72° C. for 45 seconds, followed by one cycle at 72° C. for 7minutes. Thus, the 5′ terminal of the polynucleotide sequence waselucidated. In order to subclone the 5′ portion of the polynucleotide into a pCR2.1 cloning vector, TA Cloning Kit (Invitrogen, Carlsbad,Calif.), oligonucleotides ZC 18227 (SEQ ID NO:4) and ZC17035 (SEQ IDNO:5), designed to the final 5′ terminal of the polynucleotide sequenceand the original EST clone, respectively, were used as primers. MarathoncDNA prepared from bone marrow was used as a template. Thermocyclerconditions were as follows: one cycle at 94° C. for 2 minutes; followedby thirty cycles at 94° C. for 20 seconds, 65° C. for 30 seconds, 72° C.for 30 seconds, followed by one cycle at 72° C. for 5 minutes. Theresulting PCR products were gel-purified, subcloned, and sequenced. Aconsensus sequence was generated by combining the sequence from the PCRproducts with the cDNA sequence from the EST clone.

[0217] Polymorphisms were evident in the consensus sequence at thefollowing positions: R at nucleotide positions 172 and 509, and M atnucleotide position 700; where R is G or A, and M is C or A. A versionof the consensus sequence, with a G chosen at position 172 and an A atposition 700, was joined to the original EST clone. Thus, amino acidresidue 137 of SEQ ID NO:2 is either glycine or serine.

Example 2

[0218] Tissue Distribution

[0219] Analysis of tissue distribution was performed by the Northernblotting technique using Human Multiple Tissue and Master Dot Blots(Clontech, Palo Alto, Calif.). A 134 bp probe was obtained by PCR of theoriginal EST template using primers ZC16893 (SEQ ID NO:6) and ZC16894(SEQ ID NO:7). Thermocycler conditions were as follows: one cycle at 94°C. for 2 minutes; followed by thirty cycles at 94° C. for 20 seconds,65° C. for 30 seconds, 72° C. for 30 seconds, followed by one cycle at72° C. for 2 minutes. The PCR product was random prime labeled with ³²pusing a commercially available kit (Rediprime DNA Labeling System;Amersham Corp., Arlington Heights, Ill.) according to the manufacturer'sdirection. The probe was then purified using a NucTrap® probepurification column (Stratagene, La Jolla, Calif.). ExpressHyb™Hybridization Solution (Clontech, Palo Alto, Calif.) was used forpre-hybridization and hybridization. Hybridization took place overnightat 65° C., and the blots were then washed four times in 2×SSC and 0.05%SDS at 50° C., followed by washing twice in 0.1×SSC and 0.1% SDS at 50°C., and developed. Transcripts of about 1.2 kb, 2.8 kb, 4.5 kb and 7.0kb showed very strong signals in pancreas. Longer exposure indicatesthat mRNA (of about 1.8 kb) is also present in tissues including heart,placenta, lung, skeleton muscle, kidney, spleen, prostate, smallintestine, colon, peripheral leukocyte, stomach, thyroid, spinal cord,lymph node, trachea, and bone marrow.

Example 3

[0220] Chromosomal Assignment and Placement of znssp2.

[0221] Znssp2 was mapped to chromosome 19 using the commerciallyavailable version of the “Stanford G3 Radiation Hybrid Mapping Panel”(Research Genetics, Inc., Huntsville, Ala.). The “Stanford G3 RH Panel”contains PCRable DNAs from each of 83 radiation hybrid clones of thewhole human genome, plus two control DNAs (the RM donor and the A3recipient). A publicly available WWW server(http://shgc-www.stanford.edu) allows chromosomal localization ofmarkers.

[0222] For the mapping of znssp2 with the “Stanford G3 RH Panel”, 20 μlreactions were set up in a 96-well microtiter plate used forPCR(Stratagene, La Jolla, Calif.) and used in a “RoboCycler Gradient 96”thermal cycler (Stratagene). Each of the 85 PCR reactions consisted of 2μl 10×KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., PaloAlto, Calif.), 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City,Calif.), 1 μl sense primer, ZC 19,140 (SEQ ID NO:17), 1 μl antisenseprimer, ZC 19,141 (SEQ ID NO:18), 2 μl “RediLoad” (Research Genetics,Inc., Huntsville, Ala.), 0.4 μl 50×Advantage KlenTaq Polymerase Mix(Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybridclone or control and ddH₂O for a total volume of 20 μl. The reactionswere overlaid with an equal amount of mineral oil and sealed. The PCRcycler conditions were as follows: an initial 1 cycle 5 minutedenaturation at 94° C., 35 cycles of a 45 seconds denaturation at 94°C., 45 seconds annealing at 64° C. and 1 minute AND 15 seconds extensionat 72° C., followed by a final 1 cycle extension of 7 minutes at 72° C.The reactions were separated by electrophoresis on a 2% agarose gel(Life Technologies, Gaithersburg, Md.).

[0223] The results showed linkage of mssp2 to the framework markerSHGC-33470 with a LOD score of >15 and at a distance of 3.85 cR_(—)10000from the marker.

[0224] The use of surrounding markers positions znssp2 in the 19q13region on the integrated LDB chromosome 19 map (The Genetic LocationDatabase, University of Southhampton, WWW server: http://cedar.genetics.soton.ac.uk/public_html/).

Example 4

[0225] Construct for generating znssp2h Transgenic Mice

[0226] Oligonucleotides were designed to generate a PCR fragmentcontaining a consensus Kozak sequence and the exact znssp2 human(znssp2h) coding region (nucleotides 101 to 1294 of SEQ ID NO:1). Theseoligonucleotides were designed with an PmeI site at the 5′ end and anAscI site at the 3′ end to facilitate cloning into pTg12-8, our standardtransgenic vector. PTg12-8 contains the mouse MT-1 promoter and a 5′ ratinsulin II intron upstream of the PmeI site.

[0227] A full-length clone of znssp2h was generated by the ligation: Asequencing vector containing a znssp2h DNA segment from nucleotide 1 tonucleotide 696 of SEQ ID NO:1, (with the addition of the EcoRi site atthe 5′ end) was digested from the sequencing vector as an EcoRI/SpeIfragment. Similarly, a sequencing vector containing a znssp2h DNAsegment from nucleotide 691 to 1359 of SEQ ID NO:1 was digested from thesequencing vector as a SpeI/NaeI fragment. These digested fragments wereligated to a pre-digested (EcoRI/HincII) pUC19 cloning vector. TheNaeI-HincII sites were destroyed in this ligation. A glycine residue wasused at position 137 of SEQ ID NO:2.

[0228] About one microliter of the ligation reaction was electroporatedinto DHIOB ElectroMax™ competent cells (GIBCO BRL, Gaithersburg, Md.)according to manufacturer's direction and plated onto LB platescontaining 100 μg/ml ampicillin, and incubated overnight. Colonies werepicked and grown in LB media containing 100 μg/ml ampicillin. MiniprepDNA was prepared from the picked clones and screened for the znssp2hinsert by restriction digestion with EcoRI, and subsequent agarose gelelectrophoresis.

[0229] A polymerase chain reaction using the this full-length sequenceas template (200 ng) was used to add a PmeI restriction site and a Kozaksequence (oligonucleotide ZC20336, SEQ ID NO:15) to the 5′ end of theznssp2h sequence and an AscI site to the 3′ end (oligonucleotideZC20316, SEQ ID NO:16). PCR reaction conditions were as follows: 95° C.for 5 minutes, wherein Advantage cDNA polymerase (Clontech) was added;15 cycles of 95° C. for 60 seconds, 62° C. for 60 seconds, and 72° C for90 seconds; and 72° C. for 7 minutes. PCR products were separated byagarose gel electrophoresis and purified using a QiaQuick™ (Qiagen) gelextraction kit. The isolated, 1194 bp, DNA fragment was digested withPmeI and AscI (New England Biolabs), ethanol precipitated and ligatedinto pTg12-8 that was previously digested with PmeI and AscI. ThepTg12-8 plasmid, designed for expression of a gene of interest intransgenic mice, contains an expression cassette flanked by 10 kb ofMT-1 5′ DNA and 7 kb of MT-1 3′ DNA. The expression cassette comprisesthe MT-1 promoter, the rat insulin II intron, a polylinker for theinsertion of the desired clone, and the human growth hormone poly Asequence.

[0230] About one microliter of the ligation reaction was electroporatedas described above. Colonies were picked and grown in LB mediacontaining 100 μg/ml ampicillin. Miniprep DNA was prepared from thepicked clones and screened for the znssp2h insert by restrictiondigestion with EcoRI. Maxipreps of the correct pTg12-8-znssp2h constructwere performed and digested with SalI. The SalI fragment fragmentcontaining with 5′ and 3′ flanking sequences, the MT-1 promoter, the ratinsulin II intron, znssp2h cDNA and the human growth hormone poly Asequence was prepared to be used for microinjection into fertilizedmurine oocytes.

Example 5

[0231] Cloning of the Mouse Ortholog

[0232] The human znssp2 gene was used to query the mouse EST databasefor orthologs. A cDNA clone corresponding to the human znssp2 sequencewas obtained and the deduced amino acid sequence was determined to befull-length and a murine ortholog of human znssp2 (znssp2-m). Thepolynucleotide and polypeptide sequences of the mouse ortholog are shownin SEQ ID NOs:12 and 13. The degenerate sequence for the mouse orthologis shown in SEQ ID NO: 14.

Example 6

[0233] Identification of Cells Expressing znssp2 Using in situHybridization

[0234] Human pancreas tissues were isolated and screened for znssp2expression by in situ hybridization. The human tissues prepared,sectioned and subjected to in situ hybridization included pancreasesfrom normal and pancreatitis patients. The tissues were fixed in 10%buffered formalin and blocked in paraffin using standard techniques.Tissues were sectioned at 4 to 8 microns. Tissues were prepared using astandard protocol (“Development of non-isotopic in situ hybridization”at http://dir.niehs.nih.gov/dirlep/ish.html). Briefly, tissue sectionswere deparaffinized with HistoClear (National Diagnostics, Atlanta, Ga.)and then dehydrated with ethanol. Next they were digested withProteinase K (50 μg/ml) (Boehringer Diagnostics, Indianapolis, Ind.) at37° C. for 3 to 5 minutes. This step was followed by acetylation andre-hydration of the tissues.

[0235] An in situ probe generated by PCR was designed against the humanznssp2 sequence. A set of oligos were designed to generate probes forseparate regions of the znssp2 cDNA: Oligos ZC25,177 (SEQ ID NO: 19) andZC25,232 (SEQ ID NO:20) were used to generate a 551 bp probe for znssp2.The antisense oligo from the PCR primer set also contained the workingsequence for the T7 RNA polymerase promoter to allow for easytranscription of antisense RNA probes from these PCR products. The PCRreaction conditions were as follows: 35 cycles at 94° C. for 30 sec, 45°C. for 1 min., 72° C. for 1 min with 5% Dimethyl Sulpnoxide (DMSO)(Sigma Chemical Co, MO). The PCR product was purified by Qiagen spincolumns followed by phenol/chloroform extraction and ethanolprecipitation. The probe was subsequently labeled with digoxigenin(Boehringer) or biotin (Boehringer) using an In Vitro transcriptionSystem (Promega, Madison, Wis.) as per manufacturer's instruction.

[0236] In situ hybridization was performed with a digoxigenin- orbiotin-labeled znssp2 probe (above). The probe was added to the slidesat a concentration of 1 to 5 pmol/ml for 12 to 16 hours at 55-60° C.Slides were subsequently washed in 2×SSC and 0.1×SSC at 50-55° C. Thesignals were amplified using tyramide signal amplification (TSA) (TSA,in situ indirect kit; NEN) and visualized with Vector Red substrate kit(Vector Lab) as per manufacturer's instructions. The slides were thencounter-stained with hernatoxylin (Vector Laboratories, Burlingame,Calif.).

[0237] A signal was seen in both normal and pancreatitis pancreas. Thepositive-staining cells appeared to be acinar and related cells.

[0238] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 20 <210> SEQ ID NO 1<211> LENGTH: 1532 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (101)...(1294) <400>SEQUENCE: 1 atcagaggga gctgagggag gcctgacctg aggccggcac ccggagctggcgggagccag 60 acccagagct cccgcggccg ccccttccct gggtcgggtc atg cgc tgcccc aag 115 Met Arg Cys Pro Lys 1 5 tgc ctt ctc tgc ctg tca gca ctg ctcaca ctc ctg ggc ctc aaa gtg 163 Cys Leu Leu Cys Leu Ser Ala Leu Leu ThrLeu Leu Gly Leu Lys Val 10 15 20 tac atc gag tgg aca tcc gag tcc cgg ctcagc aag gcc tac ccc agc 211 Tyr Ile Glu Trp Thr Ser Glu Ser Arg Leu SerLys Ala Tyr Pro Ser 25 30 35 cct cgg ggc acc ccg cca agc ccc acg cca gccaac cct gag ccc acc 259 Pro Arg Gly Thr Pro Pro Ser Pro Thr Pro Ala AsnPro Glu Pro Thr 40 45 50 cta cct gcc aac ctc tcc acc cgc ctg ggc cag actatc ccg ctg ccc 307 Leu Pro Ala Asn Leu Ser Thr Arg Leu Gly Gln Thr IlePro Leu Pro 55 60 65 ttt gct tac tgg aac cag cag cag tgg cgg ctg ggg tccctg ccc agt 355 Phe Ala Tyr Trp Asn Gln Gln Gln Trp Arg Leu Gly Ser LeuPro Ser 70 75 80 85 ggg gac agc act gaa acg ggg ggc tgc cag gct tgg ggggcc gcc gcc 403 Gly Asp Ser Thr Glu Thr Gly Gly Cys Gln Ala Trp Gly AlaAla Ala 90 95 100 gcc acc gag atc cct gac ttc gcc tcc tac ccc aag gacctc cgc cgc 451 Ala Thr Glu Ile Pro Asp Phe Ala Ser Tyr Pro Lys Asp LeuArg Arg 105 110 115 ttc ttg ctg tca gca gcc tgc cgg agc ttc cca cag tggctg cct gga 499 Phe Leu Leu Ser Ala Ala Cys Arg Ser Phe Pro Gln Trp LeuPro Gly 120 125 130 ggt ggt ggc rgc caa gtc tcc agc tgc tca gat act gatgtc ccc tac 547 Gly Gly Gly Xaa Gln Val Ser Ser Cys Ser Asp Thr Asp ValPro Tyr 135 140 145 ctg ctg ttg gcc gtc aag tca gaa cca ggg cgc ttt gcagaa cga cag 595 Leu Leu Leu Ala Val Lys Ser Glu Pro Gly Arg Phe Ala GluArg Gln 150 155 160 165 gcc gtg aga gag acg tgg ggc agt cca gct cca gggatc cgg ctg ctc 643 Ala Val Arg Glu Thr Trp Gly Ser Pro Ala Pro Gly IleArg Leu Leu 170 175 180 ttc ctg cta ggg tct ccg gtg ggt gag gcg ggg cctgac cta gac tca 691 Phe Leu Leu Gly Ser Pro Val Gly Glu Ala Gly Pro AspLeu Asp Ser 185 190 195 cta gtg gca tgg gag agc cgt cgc tac agt gac ctgctg ctc tgg gac 739 Leu Val Ala Trp Glu Ser Arg Arg Tyr Ser Asp Leu LeuLeu Trp Asp 200 205 210 ttc ctc gac gtc cca ttc aac cag acg ctc aaa gacctg ctg ctg ctg 787 Phe Leu Asp Val Pro Phe Asn Gln Thr Leu Lys Asp LeuLeu Leu Leu 215 220 225 gcc tgg ctg ggc cgc cac tgc ccc acc gtg agt tttgtc ttg cga gct 835 Ala Trp Leu Gly Arg His Cys Pro Thr Val Ser Phe ValLeu Arg Ala 230 235 240 245 cag gac gat gcc ttt gta cac acc cct gcc ctgctg gct cac ctg cgg 883 Gln Asp Asp Ala Phe Val His Thr Pro Ala Leu LeuAla His Leu Arg 250 255 260 gcc ctg cca cct gcc tcg gcc cga agc ctc tacctg ggt gag gtc ttt 931 Ala Leu Pro Pro Ala Ser Ala Arg Ser Leu Tyr LeuGly Glu Val Phe 265 270 275 acc cag gcc atg cct ctc cgg aag cca gga ggaccc ttc tat gtg ccc 979 Thr Gln Ala Met Pro Leu Arg Lys Pro Gly Gly ProPhe Tyr Val Pro 280 285 290 gag tcc ttc ttc gaa ggt ggc tac cca gcc tatgca agc ggg ggt ggc 1027 Glu Ser Phe Phe Glu Gly Gly Tyr Pro Ala Tyr AlaSer Gly Gly Gly 295 300 305 tac gtc att gcc ggg cgc ctg gca ccc tgg ctgctg cgg gcg gca gcc 1075 Tyr Val Ile Ala Gly Arg Leu Ala Pro Trp Leu LeuArg Ala Ala Ala 310 315 320 325 cgt gtg gca ccc ttc ccc ttt gag gac gtctac act ggc ctt tgc atc 1123 Arg Val Ala Pro Phe Pro Phe Glu Asp Val TyrThr Gly Leu Cys Ile 330 335 340 cga gcc ctg ggc ctg gtg ccc cag gcc caccca ggc ttc ctc aca gcc 1171 Arg Ala Leu Gly Leu Val Pro Gln Ala His ProGly Phe Leu Thr Ala 345 350 355 tgg cca gca gac cgc act gcg gac cac tgtgct ttc cgc aac ctg ctg 1219 Trp Pro Ala Asp Arg Thr Ala Asp His Cys AlaPhe Arg Asn Leu Leu 360 365 370 ctg gta cgg ccc ctg ggc ccc cag gcc agcatt cgg ctc tgg aaa caa 1267 Leu Val Arg Pro Leu Gly Pro Gln Ala Ser IleArg Leu Trp Lys Gln 375 380 385 ctg caa gac cca agg ctc cag tgc tgactctcattgg ggagggcgga 1314 Leu Gln Asp Pro Arg Leu Gln Cys 390 395ggtgctgacc tggccccggc cctggcctgg gcctctgggg ccggcccctg gctcagcccc 1374tccttccagg tcttgatggg agggaggagg gcccagaagc tggacaactt aagccactcc 1434ttggcctccc ccagccaggg gcctgggcag gaaagatggg gtggtggact gtttttgcct 1494actttttgtt tttgaaaaac atgcactccc cactctga 1532 <210> SEQ ID NO 2 <211>LENGTH: 397 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (137)...(137) <223> OTHERINFORMATION: Xaa is Gly or Ser <220> FEATURE: <221> NAME/KEY: VARIANT<222> LOCATION: (1)...(397) <223> OTHER INFORMATION: Xaa = Any AminoAcid <400> SEQUENCE: 2 Met Arg Cys Pro Lys Cys Leu Leu Cys Leu Ser AlaLeu Leu Thr Leu 1 5 10 15 Leu Gly Leu Lys Val Tyr Ile Glu Trp Thr SerGlu Ser Arg Leu Ser 20 25 30 Lys Ala Tyr Pro Ser Pro Arg Gly Thr Pro ProSer Pro Thr Pro Ala 35 40 45 Asn Pro Glu Pro Thr Leu Pro Ala Asn Leu SerThr Arg Leu Gly Gln 50 55 60 Thr Ile Pro Leu Pro Phe Ala Tyr Trp Asn GlnGln Gln Trp Arg Leu 65 70 75 80 Gly Ser Leu Pro Ser Gly Asp Ser Thr GluThr Gly Gly Cys Gln Ala 85 90 95 Trp Gly Ala Ala Ala Ala Thr Glu Ile ProAsp Phe Ala Ser Tyr Pro 100 105 110 Lys Asp Leu Arg Arg Phe Leu Leu SerAla Ala Cys Arg Ser Phe Pro 115 120 125 Gln Trp Leu Pro Gly Gly Gly GlyXaa Gln Val Ser Ser Cys Ser Asp 130 135 140 Thr Asp Val Pro Tyr Leu LeuLeu Ala Val Lys Ser Glu Pro Gly Arg 145 150 155 160 Phe Ala Glu Arg GlnAla Val Arg Glu Thr Trp Gly Ser Pro Ala Pro 165 170 175 Gly Ile Arg LeuLeu Phe Leu Leu Gly Ser Pro Val Gly Glu Ala Gly 180 185 190 Pro Asp LeuAsp Ser Leu Val Ala Trp Glu Ser Arg Arg Tyr Ser Asp 195 200 205 Leu LeuLeu Trp Asp Phe Leu Asp Val Pro Phe Asn Gln Thr Leu Lys 210 215 220 AspLeu Leu Leu Leu Ala Trp Leu Gly Arg His Cys Pro Thr Val Ser 225 230 235240 Phe Val Leu Arg Ala Gln Asp Asp Ala Phe Val His Thr Pro Ala Leu 245250 255 Leu Ala His Leu Arg Ala Leu Pro Pro Ala Ser Ala Arg Ser Leu Tyr260 265 270 Leu Gly Glu Val Phe Thr Gln Ala Met Pro Leu Arg Lys Pro GlyGly 275 280 285 Pro Phe Tyr Val Pro Glu Ser Phe Phe Glu Gly Gly Tyr ProAla Tyr 290 295 300 Ala Ser Gly Gly Gly Tyr Val Ile Ala Gly Arg Leu AlaPro Trp Leu 305 310 315 320 Leu Arg Ala Ala Ala Arg Val Ala Pro Phe ProPhe Glu Asp Val Tyr 325 330 335 Thr Gly Leu Cys Ile Arg Ala Leu Gly LeuVal Pro Gln Ala His Pro 340 345 350 Gly Phe Leu Thr Ala Trp Pro Ala AspArg Thr Ala Asp His Cys Ala 355 360 365 Phe Arg Asn Leu Leu Leu Val ArgPro Leu Gly Pro Gln Ala Ser Ile 370 375 380 Arg Leu Trp Lys Gln Leu GlnAsp Pro Arg Leu Gln Cys 385 390 395 <210> SEQ ID NO 3 <211> LENGTH: 1191<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Degenerate sequence <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (1)...(1191) <223> OTHER INFORMATION: n =A,T,C or G <400> SEQUENCE: 3 atgmgntgyc cnaartgyyt nytntgyytn wsngcnytnytnacnytnyt nggnytnaar 60 gtntayathg artggacnws ngarwsnmgn ytnwsnaargcntayccnws nccnmgnggn 120 acnccnccnw snccnacncc ngcnaayccn garccnacnytnccngcnaa yytnwsnacn 180 mgnytnggnc aracnathcc nytnccntty gcntaytggaaycarcarca rtggmgnytn 240 ggnwsnytnc cnwsnggnga ywsnacngar acnggnggntgycargcntg gggngcngcn 300 gcngcnacng arathccnga yttygcnwsn tayccnaargayytnmgnmg nttyytnytn 360 wsngcngcnt gymgnwsntt yccncartgg ytnccnggnggnggnggnnn ncargtnwsn 420 wsntgywsng ayacngaygt nccntayytn ytnytngcngtnaarwsnga rccnggnmgn 480 ttygcngarm gncargcngt nmgngaracn tggggnwsnccngcnccngg nathmgnytn 540 ytnttyytny tnggnwsncc ngtnggngar gcnggnccngayytngayws nytngtngcn 600 tgggarwsnm gnmgntayws ngayytnytn ytntgggayttyytngaygt nccnttyaay 660 caracnytna argayytnyt nytnytngcn tggytnggnmgncaytgycc nacngtnwsn 720 ttygtnytnm gngcncarga ygaygcntty gtncayacnccngcnytnyt ngcncayytn 780 mgngcnytnc cnccngcnws ngcnmgnwsn ytntayytnggngargtntt yacncargcn 840 atgccnytnm gnaarccngg nggnccntty taygtnccngarwsnttytt ygarggnggn 900 tayccngcnt aygcnwsngg nggnggntay gtnathgcnggnmgnytngc nccntggytn 960 ytnmgngcng cngcnmgngt ngcnccntty ccnttygargaygtntayac nggnytntgy 1020 athmgngcny tnggnytngt nccncargcn cayccnggnttyytnacngc ntggccngcn 1080 gaymgnacng cngaycaytg ygcnttymgn aayytnytnytngtnmgncc nytnggnccn 1140 cargcnwsna thmgnytntg gaarcarytn cargayccnmgnytncartg y 1191 <210> SEQ ID NO 4 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer, ZC18227 <400> SEQUENCE: 4atcagaggga gctgagggag gc 22 <210> SEQ ID NO 5 <211> LENGTH: 25 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer, ZC17035 <400> SEQUENCE: 5ggtctttgag cgtctggttg aatgg 25 <210> SEQ ID NO 6 <211> LENGTH: 25 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer, ZC16893 <400> SEQUENCE: 6tacagtgacc tgctgctctg ggact 25 <210> SEQ ID NO 7 <211> LENGTH: 25 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer, ZC16894 <400> SEQUENCE: 7cctgagctcg caagacaaaa ctcac 25 <210> SEQ ID NO 8 <211> LENGTH: 23 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer, ZC9719 <400> SEQUENCE: 8 actcactatagggctcgagc gcc 23 <210> SEQ ID NO 9 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer, ZC17035 <400> SEQUENCE: 9ggtctttgag cgtctggttg aatgg 25 <210> SEQ ID NO 10 <211> LENGTH: 27 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer, ZC9739 <400> SEQUENCE: 10ccatcctaat acgactcact atagggc 27 <210> SEQ ID NO 11 <211> LENGTH: 25<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Oligonucleotide primer, ZC17036 <400> SEQUENCE: 11gaggaagtcc cagagcagca ggtca 25 <210> SEQ ID NO 12 <211> LENGTH: 1446<212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (107)...(1273) <400> SEQUENCE: 12gcacagctgc gaggagggag tccggcaggg ctttacccga ggacccccag agctggcgga 60agctggaccc agagccccac tggtggccct ttccctgggc cgggtc atg cgt tgc 115 MetArg Cys 1 cgc aag tgc cag ctc tgc ctg tca gca ctg ctc aca ctc ctg ggcctc 163 Arg Lys Cys Gln Leu Cys Leu Ser Ala Leu Leu Thr Leu Leu Gly Leu5 10 15 aaa gta tac atc gag tgg aca tcc gag tcc tgg ctt aaa aag gct gaa211 Lys Val Tyr Ile Glu Trp Thr Ser Glu Ser Trp Leu Lys Lys Ala Glu 2025 30 35 ccc cgg ggc gct ctg ccc agt ccc aca cca ccc aat gct gag ccc act259 Pro Arg Gly Ala Leu Pro Ser Pro Thr Pro Pro Asn Ala Glu Pro Thr 4045 50 ctg ccc acc aac ctc tca gca cgc ctg ggt cag act ggc cca ctg tcc307 Leu Pro Thr Asn Leu Ser Ala Arg Leu Gly Gln Thr Gly Pro Leu Ser 5560 65 tct gct tac tgg aac cag cag cag cgg cag ctg gga gtc ctg ccc agt355 Ser Ala Tyr Trp Asn Gln Gln Gln Arg Gln Leu Gly Val Leu Pro Ser 7075 80 acg gac tgt cag act tgg ggg act gtt gct gcc tcg gag atc ttg gac403 Thr Asp Cys Gln Thr Trp Gly Thr Val Ala Ala Ser Glu Ile Leu Asp 8590 95 ttc atc ctg tac ccc cag gag ctt cgg cgc ttc ttg ctg tcg gcg gcc451 Phe Ile Leu Tyr Pro Gln Glu Leu Arg Arg Phe Leu Leu Ser Ala Ala 100105 110 115 tgt agg agc ttt cca cta tgg ctg cct gca gga gaa ggc agc cctgtg 499 Cys Arg Ser Phe Pro Leu Trp Leu Pro Ala Gly Glu Gly Ser Pro Val120 125 130 gcc agc tgc tct gat aag gat gta ccc tac ttg cta ctg gct gtcaaa 547 Ala Ser Cys Ser Asp Lys Asp Val Pro Tyr Leu Leu Leu Ala Val Lys135 140 145 tca gaa cca gga cac ttt gca gca cgg cag gct gtg agg gag acctgg 595 Ser Glu Pro Gly His Phe Ala Ala Arg Gln Ala Val Arg Glu Thr Trp150 155 160 ggc agc cca gtt gct ggg acc cgg ttg ctc ttc ctg ctg ggg tccccc 643 Gly Ser Pro Val Ala Gly Thr Arg Leu Leu Phe Leu Leu Gly Ser Pro165 170 175 cta gga atg ggg ggg cct gac tta aga tca ctg gtg acg tgg gaaagc 691 Leu Gly Met Gly Gly Pro Asp Leu Arg Ser Leu Val Thr Trp Glu Ser180 185 190 195 cgg cgc tat ggt gac cta ctg ctc tgg gac ttc ctg gat gttccc tac 739 Arg Arg Tyr Gly Asp Leu Leu Leu Trp Asp Phe Leu Asp Val ProTyr 200 205 210 aac cgg aca ctc aag gac ctg ctg ctg ctg acc tgg ctg agccac cac 787 Asn Arg Thr Leu Lys Asp Leu Leu Leu Leu Thr Trp Leu Ser HisHis 215 220 225 tgc ccc gat gtc aat ttt gtc ctg cag gtt cag gat gat gccttt gtg 835 Cys Pro Asp Val Asn Phe Val Leu Gln Val Gln Asp Asp Ala PheVal 230 235 240 cac atc cca gcc cta ctg gag cac ctg cag act ctg cca cccacc tgg 883 His Ile Pro Ala Leu Leu Glu His Leu Gln Thr Leu Pro Pro ThrTrp 245 250 255 gcc cgc agc ctc tac ctg ggt gag atc ttc acc cag gcc aaaccg ctc 931 Ala Arg Ser Leu Tyr Leu Gly Glu Ile Phe Thr Gln Ala Lys ProLeu 260 265 270 275 cgc aag ccc gga gga ccc ttc tat gtg ccg aag acc ttcttt gaa ggg 979 Arg Lys Pro Gly Gly Pro Phe Tyr Val Pro Lys Thr Phe PheGlu Gly 280 285 290 gac tat cca gcc tat gcg agt gga ggt ggc tat gta atctca gga cgc 1027 Asp Tyr Pro Ala Tyr Ala Ser Gly Gly Gly Tyr Val Ile SerGly Arg 295 300 305 ctg gca ccc tgg ctg ctg cag gcg gca gct cgc gtg gcaccc ttc ccc 1075 Leu Ala Pro Trp Leu Leu Gln Ala Ala Ala Arg Val Ala ProPhe Pro 310 315 320 ttt gat gat gtc tac act ggc ttc tgc ttc cgt gcc ctgggc tta gca 1123 Phe Asp Asp Val Tyr Thr Gly Phe Cys Phe Arg Ala Leu GlyLeu Ala 325 330 335 ccc cgt gcc cat cca ggc ttc ctc aca gcc tgg cca gcagaa cgt acc 1171 Pro Arg Ala His Pro Gly Phe Leu Thr Ala Trp Pro Ala GluArg Thr 340 345 350 355 agg gac ccc tgc gcc gtg cga ggc ctg ctc ttg gtgcat cca gtc agc 1219 Arg Asp Pro Cys Ala Val Arg Gly Leu Leu Leu Val HisPro Val Ser 360 365 370 cct cag gac acc att tgg ctc tgg aga cat ctg tgggtc cca gag ctc 1267 Pro Gln Asp Thr Ile Trp Leu Trp Arg His Leu Trp ValPro Glu Leu 375 380 385 cag tgc tgaccggcag agacaagctg gggtgggtgggtgctgacct ggcctgagtc 1323 Gln Cys tctcctagag acaagctggg gtgggtggggctgacctggc ctgagtctct cctaaaccct 1383 tcctagccaa ggtggcagac tgtgtttatctactttatgg ttttgaaaaa tgtgtccttc 1443 cta 1446 <210> SEQ ID NO 13 <211>LENGTH: 389 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE:13 Met Arg Cys Arg Lys Cys Gln Leu Cys Leu Ser Ala Leu Leu Thr Leu 1 510 15 Leu Gly Leu Lys Val Tyr Ile Glu Trp Thr Ser Glu Ser Trp Leu Lys 2025 30 Lys Ala Glu Pro Arg Gly Ala Leu Pro Ser Pro Thr Pro Pro Asn Ala 3540 45 Glu Pro Thr Leu Pro Thr Asn Leu Ser Ala Arg Leu Gly Gln Thr Gly 5055 60 Pro Leu Ser Ser Ala Tyr Trp Asn Gln Gln Gln Arg Gln Leu Gly Val 6570 75 80 Leu Pro Ser Thr Asp Cys Gln Thr Trp Gly Thr Val Ala Ala Ser Glu85 90 95 Ile Leu Asp Phe Ile Leu Tyr Pro Gln Glu Leu Arg Arg Phe Leu Leu100 105 110 Ser Ala Ala Cys Arg Ser Phe Pro Leu Trp Leu Pro Ala Gly GluGly 115 120 125 Ser Pro Val Ala Ser Cys Ser Asp Lys Asp Val Pro Tyr LeuLeu Leu 130 135 140 Ala Val Lys Ser Glu Pro Gly His Phe Ala Ala Arg GlnAla Val Arg 145 150 155 160 Glu Thr Trp Gly Ser Pro Val Ala Gly Thr ArgLeu Leu Phe Leu Leu 165 170 175 Gly Ser Pro Leu Gly Met Gly Gly Pro AspLeu Arg Ser Leu Val Thr 180 185 190 Trp Glu Ser Arg Arg Tyr Gly Asp LeuLeu Leu Trp Asp Phe Leu Asp 195 200 205 Val Pro Tyr Asn Arg Thr Leu LysAsp Leu Leu Leu Leu Thr Trp Leu 210 215 220 Ser His His Cys Pro Asp ValAsn Phe Val Leu Gln Val Gln Asp Asp 225 230 235 240 Ala Phe Val His IlePro Ala Leu Leu Glu His Leu Gln Thr Leu Pro 245 250 255 Pro Thr Trp AlaArg Ser Leu Tyr Leu Gly Glu Ile Phe Thr Gln Ala 260 265 270 Lys Pro LeuArg Lys Pro Gly Gly Pro Phe Tyr Val Pro Lys Thr Phe 275 280 285 Phe GluGly Asp Tyr Pro Ala Tyr Ala Ser Gly Gly Gly Tyr Val Ile 290 295 300 SerGly Arg Leu Ala Pro Trp Leu Leu Gln Ala Ala Ala Arg Val Ala 305 310 315320 Pro Phe Pro Phe Asp Asp Val Tyr Thr Gly Phe Cys Phe Arg Ala Leu 325330 335 Gly Leu Ala Pro Arg Ala His Pro Gly Phe Leu Thr Ala Trp Pro Ala340 345 350 Glu Arg Thr Arg Asp Pro Cys Ala Val Arg Gly Leu Leu Leu ValHis 355 360 365 Pro Val Ser Pro Gln Asp Thr Ile Trp Leu Trp Arg His LeuTrp Val 370 375 380 Pro Glu Leu Gln Cys 385 <210> SEQ ID NO 14 <211>LENGTH: 1167 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: degenerate sequence <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1167) <223> OTHERINFORMATION: n = A,T,C or G <400> SEQUENCE: 14 atgmgntgym gnaartgycarytntgyytn wsngcnytny tnacnytnyt nggnytnaar 60 gtntayathg artggacnwsngarwsntgg ytnaaraarg cngarccnmg nggngcnytn 120 ccnwsnccna cnccnccnaaygcngarccn acnytnccna cnaayytnws ngcnmgnytn 180 ggncaracng gnccnytnwsnwsngcntay tggaaycarc arcarmgnca rytnggngtn 240 ytnccnwsna cngaytgycaracntggggn acngtngcng cnwsngarat hytngaytty 300 athytntayc cncargarytnmgnmgntty ytnytnwsng cngcntgymg nwsnttyccn 360 ytntggytnc cngcnggngarggnwsnccn gtngcnwsnt gywsngayaa rgaygtnccn 420 tayytnytny tngcngtnaarwsngarccn ggncayttyg cngcnmgnca rgcngtnmgn 480 garacntggg gnwsnccngtngcnggnacn mgnytnytnt tyytnytngg nwsnccnytn 540 ggnatgggng gnccngayytnmgnwsnytn gtnacntggg arwsnmgnmg ntayggngay 600 ytnytnytnt gggayttyytngaygtnccn tayaaymgna cnytnaarga yytnytnytn 660 ytnacntggy tnwsncaycaytgyccngay gtnaayttyg tnytncargt ncargaygay 720 gcnttygtnc ayathccngcnytnytngar cayytncara cnytnccncc nacntgggcn 780 mgnwsnytnt ayytnggngarathttyacn cargcnaarc cnytnmgnaa rccnggnggn 840 ccnttytayg tnccnaaracnttyttygar ggngaytayc cngcntaygc nwsnggnggn 900 ggntaygtna thwsnggnmgnytngcnccn tggytnytnc argcngcngc nmgngtngcn 960 ccnttyccnt tygaygaygtntayacnggn ttytgyttym gngcnytngg nytngcnccn 1020 mgngcncayc cnggnttyytnacngcntgg ccngcngarm gnacnmgnga yccntgygcn 1080 gtnmgnggny tnytnytngtncayccngtn wsnccncarg ayacnathtg gytntggmgn 1140 cayytntggg tnccngarytncartgy 1167 <210> SEQ ID O 15 <211> LENGTH: 35 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:oligonucleotide ZC20336 <400> SEQUENCE: 15 gcgagcgttt aaacgccaccatgcgctgcc ccaag 35 <210> SEQ ID NO 16 <211> LENGTH: 32 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: oligonucleotide ZC20316 <400> SEQUENCE: 16 cgtatcggcgcgcctcagca ctggagcctt gg 32 <210> SEQ ID NO 17 <211> LENGTH: 18 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer, 19140 <400> SEQUENCE: 17 gtgcccgagtccttcttc 18 <210> SEQ ID NO 18 <211> LENGTH: 18 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:oligonucleotide primer, ZC19141 <400> SEQUENCE: 18 gcaaaggcca gtgtagac18 <210> SEQ ID NO 19 <211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide, ZC25177 <400> SEQUENCE: 19 atgcattaac cctcactaaagggcatgcct ctccggaagc cag 43 <210> SEQ ID NO 20 <211> LENGTH: 43 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide, ZC25232 <400> SEQUENCE: 20 taatacgactcactataggg aggcaaaaac agtccaccac ccc 43

What is claimed is:
 1. An isolated polypeptide comprising residues 148to 397 of SEQ ID NO:2.
 2. The isolated polypeptide according to claim 1wherein the polypeptide comprises residues 19 to 397 of SEQ ID NO:2. 3.The isolated polypeptide according to claim 2 wherein the polypeptidecomprises residues 1 to 397 of SEQ ID NO:2.
 4. An isolated polypeptideselected from the group consisting of: a) a polypeptide comprisingresidues 1 to 18 of SEQ ID NO:2; b) a polypeptide comprising residues 19to 147 of SEQ ID NO:2; c) a polypeptide comprising residues 148 to 397of SEQ ID NO:2; d) a polypeptide comprising residues 19 to 397 of SEQ IDNO:2; and e) a polypeptide comprising residues 1 to 397 of SEQ ID NO:2.5. An isolated polynucleotide encoding a polypeptide wherein thepolypeptide comprises residues 148 to 397 of SEQ ID NO:2.
 6. Theisolated polynucleotide according to claim 5, wherein the polypeptidemolecule comprises residues 19 to 397 of SEQ ID NO:2.
 7. The isolatedpolynucleotide according to claim 5, wherein the polypeptide moleculecomprises residues 1 to 397 of SEQ ID NO:2.
 8. An isolatedpolynucleotide encoding a polypeptide molecule wherein the polypeptideis selected from the group consisting of: a) a polypeptide comprisingresidues 1 to 18 of SEQ ID NO:2; b) a polypeptide comprising residues 19to 147 of SEQ ID NO:2; c) a polypeptide comprising residues 148 to 397of SEQ ID NO:2; d) a polypeptide comprising residues 19 to 397 of SEQ IDNO:2; and e) a polypeptide comprising residues 1 to 397 of SEQ ID NO:2.9. An expression vector comprising the following operably linkedelements: a transcription promoter; a DNA segment wherein the DNAsegment is a polynucleotide encoding the polypeptide of claim 1; and atranscription terminator.
 10. The expression vector according to claim 9wherein the DNA segment contains an affinity tag.
 11. A cultured cellinto which has been introduced an expression vector according to claim9, wherein said cell expresses the polypeptide encoded by the DNAsegment.
 12. A method of producing a polypeptide comprising culturing acell according to claim 11, whereby said cell expresses the polypeptideencoded by the DNA segment; and recovering the polypeptide.
 13. A methodof producing an antibody comprising the following steps in order:inoculating an animal with a polypeptide selected from the groupconsisting of: a) polypeptide comprising residues 1 to 18 of SEQ IDNO:2; b) a polypeptide comprising residues 19 to 147 of SEQ ID NO:2; c)a polypeptide comprising residues 148 to 397 of SEQ ID NO:2; d) apolypeptide comprising residues 19 to 397 of SEQ ID NO:2; and e) apolypeptide comprising residues 1 to 397 of SEQ ID NO:2. wherein thepolypeptide elicits an immune response in the animal to produce theantibody; and isolating the antibody from the animal.
 14. An antibodyproduced by the method of claim 13, which binds to a residues 1 to 397of SEQ ID NO:2.
 15. The antibody of claim 14, wherein the antibody is amonoclonal antibody.
 16. An antibody which specifically binds to apolypeptide of claim
 3. 17. A method of producing an antibody comprisingthe following steps in order: inoculating an animal with an epitopebearing portion of a polypeptide wherein the epitope bearing portion isselected from the group consisting of: a) a polypeptide consisting ofresidues 1 to 6 of SEQ ID NO:2; b) a polypeptide consisting of residues26 to 54 of SEQ ID NO:2; c) a polypeptide consisting of residues 82 to94 of SEQ ID NO:2; d) a polypeptide consisting of residues 110 to 117 ofSEQ ID NO:2; e) a polypeptide consisting of residues 110 to 127 of SEQID NO:2; f) a polypeptide consisting of residues 122 to 127 of SEQ IDNO:2; g) a polypeptide consisting of residues 122 to 136 of SEQ ID NO:2;h) a polypeptide consisting of residues 131 to 136 of SEQ ID NO:2; i) apolypeptide consisting of residues 131 to 146 of SEQ ID NO:2; j) apolypeptide consisting of residues 139 to 146 of SEQ ID NO:2; k) apolypeptide consisting of residues 154 to 177 of SEQ ID NO:2; l) apolypeptide consisting of residues 187 to 197 of SEQ ID NO:2; m) apolypeptide consisting of residues 187 to 207 of SEQ ID NO:2; n) apolypeptide consisting of residues 202 to 207 of SEQ ID NO:2; o) apolypeptide consisting of residues 282 to 289 of SEQ ID NO:2; p) apolypeptide consisting of residues 282 to 301 of SEQ ID NO:2; q) apolypeptide consisting of residues 295 to 301 of SEQ ID NO:2; r) apolypeptide consisting of residues 358 to 365 of SEQ ID NO:2; s) apolypeptide consisting of residues 358 to 397 of SEQ ID NO:2; and t) apolypeptide consisting of residues 387 to 397 of SEQ ID NO:2; whereinthe polypeptide elicits an immune response in the animal to produce theantibody; and isolating the antibody from the animal.
 18. An antibodyproduced by the method of claim 17, which binds to a residues 1 to 397of SEQ ID NO:2.
 19. The antibody of claim 18, wherein the antibody is amonoclonal antibody.
 20. A method for modulating cell-cell interactionsby combining the polypeptide according to claim 1, with cells in vivoand in vitro.
 21. A method for modulating cell-cell interactionsaccording to claim 20, whereby the cells are derived from tissuesselected from the group consisting of: a) tissues from pancreas; b)tissues from colon; c) tissues from small intestine; d) tissues frombladder; e) tissues from prostate; f) tissues from myometrium; and g)tissues from breast.
 22. A method for modulating glycoprotein andglycolipid biosynthesis by combining the polypeptide according to claim1, with cells in vivo and in vitro.
 23. A method for modulatingcell-cell interactions according to claim 22, whereby the cells arederived from tissues selected from the group consisting of: a) tissuesfrom pancreas; b) tissues from colon; c) tissues from small intestine;d) tissues from bladder; e) tissues from prostate; f) tissues frommyometrium; and g) tissues from breast.
 24. A method of detecting amolecule which binds to a polypeptide comprising contacting thepolypeptide with a test sample containing the molecule wherein thepolypeptide comprises residues 148 to 397 of SEQ ID NO:2 and whereby themolecule binds the polypeptide.